HIV immunoassays using synthetic envelope polypeptides

ABSTRACT

Polynucleotide sequences are provided for the diagnosis of the presence of retroviral infection in a human host associated with lymphadenopathy syndrome and/or acquired immune deficiency syndrome, for expression of polypeptides and use of the polypeptides to prepare antibodies, where both the polypeptides and antibodies may be employed as diagnostic reagents or in therapy, e.g., vaccines and passive immunization. The sequences provide detection of the viral infectious agents associated with the indicated syndromes and can be used for expression of antigenic polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a: Continuation of application Ser. No. 07/931,154,filed Aug. 17, 1992 now abandoned.

This application is a continuation-in-part of U.S. patent applicationSer. No. 06/773,447, filed 6 Sep. 1985, which is a continuation-in-partof U.S. patent application Ser. No. 06/696,534, filed 30 Jan. 1985, nowabandoned, which is a continuation-in-part of U.S. patent applicationSer. No. 06/667,501, filed 31 Oct. 1984, now abandoned. The disclosuresof the above application are incorporated herein by reference.

TECHNICAL FIELD

The present invention is directed to nucleotide sequences, such as DNA,encoding human immunodeficiency virus polypeptides, the use of suchnucleotide sequences in diagnostic procedures and in the production ofrecombinant protein, as well as the use of such proteins in diagnostic,prophylactic, and therapeutic applications.

BACKGROUND OF THE INVENTION

Acquired immune deficiency syndrome (AIDS) is now recognized as one ofthe greatest health threats facing modern medicine. There is, as yet, nocure for this almost invariably fatal disease. This state of affairs hasmade the prevention of the disease an extremely high priority in themedical community. An individual who is infected with humanimmunodeficiency virus (HIV), the etiologic agent of AIDS, can transmitthe disease, and yet remain asymptomatic for many years. The ability toaccurately screen large numbers of asymptomatic individuals (e.g.,healthy appearing blood donors) for HIV infection is of greatimportance. Furthermore, the development of a vaccine would beparticularly desirable, since it would afford some protection againsttransmission of AIDS by individuals who either are not detected by adiagnostic test, or evade such a test.

In 1983-1984, three groups independently identified the suspectedetiological agent of AIDS. See, e.g., Barre-Sinoussi et al. (1983)Science 220:868-871; Montagnier et al., in Human T-Cell Leukemia Viruses(Gallo, Essex & Gross, eds., 1984); Vilmer et al. (1984) The Lancet1:753; Popovic et al. (1984) Science 224:497-500; Levy et al. (1984)Science 225:840-842. These isolates were variously calledlymphadenopathy-associated virus (LAV), human T-cell lymphotropic virustype III (HTLV-III), or AIDS-associated retrovirus (ARV). All of theseisolates are strains of the same virus, and were later collectivelynamed human immunodeficiency virus (HIV). With the isolation of arelated AIDS-causing virus, the strains originally called HIV are nowtermed HIV-1 and the related virus is called HIV-2. See, e.g., Guyaderet al. (1987) Nature 326:662-669; Brun-Vezinet et al. (1986) Science233:343-346; Clavel et al. (1986) Nature 324:691-695.

Initially, HIV was propagated in culture in human mitogen-activated Tcells. This method, however, could not produce the large quantities ofvirus required for serology assays on the scale required to protectpublic health and safety. It was not until immortalized cell linescapable of becoming chronically infected in vitro were discovered thatHIV could be produced in any substantial quantities. See, e.g.,Montagnier et al. (1984) Science 225:63-66; Levy et al., supra; Popovicet al., supra. The ability to grow the virus in culture led to thedevelopment of immunoassays for the detection of anti-HIV antibodies inthe blood of patients suspected of having been infected, as well as forscreening blood donors. See, e.g., Schupbach et al. (1984) Science224:503-505; Sarngadharan et al. (1984) Science 224:506-508; Feorino etal. (1984) Science 225:69-72; Kalyanaraman et al. (1984) Science225:321-323; Culliton et al. (1984) Science 226:1128-1131; Groopman etal. (1984) Science 226:447-449; Ho et al. (1984) Science 226:451-453,U.S. Pat. No. 4,520,113.

Due to the great hazard of cultivating HIV in vitro, the number offacilities and individuals capable of working with the virus isnecessarily limited. Furthermore, while tissue culture may provide viralpolypeptides suitable for use in diagnostic assays, it is highlyundesirable to employ polypeptides produced by tissue culture in vaccinecompositions due to the risk of infectivity posed by live, intact virus.

While production of viral polypeptides by recombinant means could beconsidered to be a solution to the problems described above, theproduction of recombinant proteins was not possible prior to the presentinvention. For example, HIV nucleotide sequences were not available andsequenced so as to enable the production of recombinant proteins. Evenmore importantly, it was unknown whether recombinantly produced viralprotein would be sufficiently similar in antigenic properties to nativeHIV polypeptides so as to be generally useful in diagnostic assays orvaccine production. In addition, homology between the genome of HIV andhuman T-cell leukemia virus type I and type II (HTLV-I and -II) had beenreported. See, e.g., Arya et al. (1984) Science 225:927-930. Thus, itwas unclear that sufficiently unique epitopes of HIV could be producedby recombinant means to distinguish HIV from HTLV-I or HTLV-II.Furthermore, it was unclear prior to the present invention whether thevarious HIV isolates possessed sufficiently related epitopes so that arecombinant polypeptide based on one isolate could be useful in ageneral diagnostic assay or vaccine composition.

Prior to the present invention, therefore, recombinant HIV polypeptidescould not be produced and it was not clear that such polypeptides wouldbe generally useful in diagnostic, prophylactic, or therapeutic methodsor products.

SUMMARY OF THE INVENTION

Nucleotide sequences and expression of nucleotide sequences are providedfor detecting the presence of complementary sequences associated with aretroviral etiologic agent (HIV, e.g., HIV-1 or -2) for lymphadenopathysyndrome (LAS), acquired immune deficiency syndrome (AIDS) orAIDS-related complex (ARC), and for producing polypeptides. Thesingle-stranded sequences are at least 20, more usually of at leastabout 50 nucleotides in length, and may find use as probes. Thedouble-stranded sequences may find use as genes coding for expression ofpolypeptides, either fragments or complete polypeptides expressed by thevirus or fused proteins, for use in diagnosis of HIV infection orevaluating stage of infection, the production of antibodies to HIV, andthe production of vaccines. Based on the nucleotide sequences, syntheticpeptides may also be prepared.

Specific aspects of the invention include:

1. A DNA construct comprising a replication system recognized by aunicellular microorganism and a DNA sequence coding for at least 20 bpof a human immunodeficiency virus (HIV) genome, said replication systembeing a non-HIV replication system;

2. A DNA construct comprising a replication system recognized by aunicellular microorganism and a DNA sequence of at least about 21 bphaving an open reading frame and having a sequence substantiallycomplementary to a sequence found in the gag, env, or pol region of anHIV, coding for a polypeptide which is immunologicallynon-cross-reactive with HTLV-I and HTVL-II, and reactive with an HIV;

3. A restriction endonuclease fragment of at least about 1.5 kbp derivedfrom restriction enzyme digestion by at least one restrictionendonuclease of a DNA sequence coding for an HIV of the class HIV-1;

4. A DNA sequence comprising a fragment of at least about 20 bp, whereinthe strands are complementary to a restriction endonuclease fragmentdescribed in 3 above, said sequence duplexing with an HIV nucleic acidsequence and not duplexing with HTLV-I or HTLV-II under comparableselective hybridization conditions;

5. A method for detecting the presence of an HIV nucleic acid sequencepresent in a nucleic acid sample obtained from a physiological sample,which comprises:

(a) combining said nucleic acid sample with a single-stranded nucleicacid sequence of at least about 20 bases complementary to a sequence insaid HIV and non-cross-reactive with HTLV-I and -II under conditions ofpredetermined stringency for hybridization; and

(b) detecting duplex formation between said DNA sequence and nucleicacid present in said sample;

6. A method for cloning DNA specific for an HIV, which comprises growinga unicellular microorganism containing the above-described DNAconstruct, whereby said DNA sequence is replicated;

7. A method for producing an expression product of HIV which comprises:

(a) transforming a unicellular microorganism host with a DNA constructhaving transcriptional and translational initiation and terminationregulatory signals functional in said host and an HIV DNA sequence of atleast 21 bp having an open reading frame and under the regulatorycontrol of said signals; and

(b) growing said host in a nutrient medium, whereby said expressionproduct is produced;

8. A method for producing an expression product of HIV which comprisesgrowing mammalian host cells having a DNA construct comprisingtranscriptional and translational initiation and termination regulatorysignals functional in said host cells and a DNA sequence of at least 21bp and less than the whole HIV genome, said sequence having an openreading frame and an initiation codon at its 5′-terminus and under thetranscriptional and translational control of said regulatory signals,whereby a polypeptide encoded by said sequence is expressed;

9. A method of detecting antibodies to HIV in a sample suspected ofcontaining said antibodies comprising:

-   -   (a) providing a support with at least one antigenic recombinant        HIV polypeptide bound thereto;    -   (b) contacting said sample with said support-bound polypeptide;    -   (c) washing the support;    -   (d) contacting the support with labeled antibody to human        immunoglobulin; and    -   (e) detecting the presence of said antibodies to HIV on said        support via said label;

10. Recombinant HIV polypeptides including, but not limited to:

-   -   (a) p16gag;    -   (b) p25gag;    -   (c) an env polypeptide;    -   (d) p31pol;    -   (e) a fusion protein of p16gag and p25gag;    -   (f) a fusion protein of a gag polypeptide and an env        polypeptide;    -   (g) a fusion protein comprising an env polypeptide;    -   (h) a fusion protein comprising p31pol;    -   (i) gp120env;    -   (j) gp41env;    -   (k) a fusion protein comprising env-5b; and    -   (l) reverse transcriptase.

11. An article of manufacture for use in an assay for anti-HIVantibodies comprising at least one of the above-described HIVpolypeptides bound to a solid support.

12. A vaccine composition, and a method of producing antibodies in amammal comprising administering to said mammal said vaccine compositionwherein the vaccine composition comprises an antigenically effectiveamount of a recombinant HIV polypeptide.

Other embodiments will also be apparent from the description below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a restriction map of proviral DNA from HIV strain ARV-2.

FIGS. 2 and 3 are restriction maps of recombinant λ phages containingARV-2 sequences.

FIG. 4 is a complete nucleotide sequence of ARV-2, derived from partialsequences of several ARV clones. Corresponding amino acid sequences areindicated for the open reading frames of the individual genes.

FIG. 5 is the nucleotide sequence of ARV-2(9B). The amino acid sequencesfor the products of the gag, pol, and env genes are indicated. The U3,R, and U5 regions of the LTRs are also designated. The cap site isposition +1. The nucleotides at the beginning of each line are numbered,and the amino acids at the end of each line are indicated. FIG. 5 hereinshows the same sequence as that in FIG. 5 of both U.S. Ser. No. 773,447(filed 6 Sep. 1985) and U.S. Ser. No. 696,534 (filed 30 Jan. 1985), thenucleotides in the figure of the earlier applications being numberedfrom the beginning of the integrated sequences.

FIG. 6 is a flow diagram showing the procedure for making the plasmid ofpSVi-7c/env, an expression vector for ARV-2 env gene.

FIG. 7 is a flow diagram showing the procedures for making the plasmidspGAG25-10 and pGAG41-10.

FIG. 8 is the nucleotide sequence of the p25gag gene cloned in plasmidpGAG25-10 and the amino acid sequence encoded by that gene.

FIG. 9 is the coding strand of the nucleotide sequence cloned inpGAG41-10 for producing the fusion protein p41gag and the correspondingamino acid.

FIG. 10 is a nucleotide sequence coding for p16gag protein that wascloned into plasmid ptac5 to make an expression plasmid for producingp16gag in bacteria.

FIG. 11 is a nucleotide sequence that encodes ARV-2 env protein that wasused to prepare plasmid pDPC303.

FIG. 12 is a nucleotide sequence that encodes ARV-2 p31 protein and iscontained in plasmid pTP31.

FIG. 13 is a map of the ARV env gene showing the regions env-1, env-2,env-3, env-4, and env-5.

FIG. 14 is a restriction map of plasmid pDM15, which was used toconstruct S. cerevisiae strain JSC302.

FIG. 15 is the synthetic nucleotide sequence env-5b, which encodes theamino acid sequence of the ARV env-5 region.

FIG. 16 is the results of an indirect ELISA in which an AIDS patient'sserum (⋄) was titrated against microtiter plates coated with recombinantpolypeptides from env regions. A pool of serum samples from random blooddonors was used as a control (∘). Panel A shows the results forpurified, recombinant env-2. Panel B shows the results with purified,recombinant env-5b. The insert in each panel shows a Coomassie-stainedgel (lane 1) and an immunoblot with the AIDS patient's serum (lane 2) ofthe purified antigens used in these ELISAS.

FIG. 17 shows the results of an ELISA, employing recombinant env-2 (toppanel) and env-5b (bottom panel) polypeptides, run on seronegative blooddonors.

FIG. 18 shows the results of an ELISA, employing recombinant env-2 (toppanel) and env-5b (bottom panel) polypeptides, run on HIV seropositivepatients, including those diagnosed as having AIDS or AIDS-relatedcomplex (ARC), as well as those having contacts with AIDS patients.

FIG. 19 shows the results of ELISAs used to measure antibody titers inthe AIDS seropositive patients of FIG. 18.

FIG. 20 is a flow diagram showing the procedure for making plasmidpAB24/RT4, an expression vector for HIV reverse transcriptase.

FIG. 21 is a flow diagram showing the construction of pCl/1-p25-ADH-GAP,a yeast expression vector for p25gag.

FIG. 22 shows the DNA and amino acid sequences of the p25gag structuralregion in pCl/1-p25-ADH-GAP.

FIG. 23 is a flow diagram showing the construction ofpCl/1-pSP31-ADH-GAP (pCl/1-pSP31-GAP-ADH2), a yeast expression vectorfor a SOD/p31pol fusion protein.

FIG. 24 shows the DNA and amino acid sequences of the SOD/p31polstructural region in pCl/1-pSP31-ADH-GAP.

FIG. 25 is a flow diagram showing the construction of pSOD/env5b frompSODCF2 and a synthetic env-5b sequence.

FIG. 26 shows the nucleotide sequence and putative amino acid sequenceof the SOD/env-4 fusion construct in pBS24/SOD-SFenv4.

FIG. 27 is a restriction map for yeast shuttle vector pAB24.

FIG. 28 is a restriction map for yeast expression vector pAB-GAP-env2.

FIG. 29 is a restriction map of pCMV6a.

FIG. 30 is an immunoblot performed with AIDS patient serum on env-1(lanes A, B), env-2 (lanes C, D) and env-3 (lanes E, F). Lanes A, C andE are immunoblots with normal sera, while lanes B, D and F areimmunoblots with serum from an AIDS patient.

FIG. 31 shows an ELISA survey for p31 antibodies. Panel (a) shows theresults for random, normal blood donors. Panel (b) shows the results forvirus-seropositive individuals. The shaded bars are for sera that scorednegative in the virus immunoblot assays.

MODES FOR CARRYING OUT THE INVENTION

Nucleotide sequences are provided which are at least in part specificfor sequences present in HIV retroviruses, which are the etiologicalagent of AIDS. HIV is an art-recognized family of viruses, e.g., HIV-1and HIV-2. The original isolates of these viruses were variably referredto as lymphadenopathy virus (LAV) [Barre-Sinoussi et al. (1983) Science220:868-871], human T-cell lymphotropic virus-III (HTLV-III) [Popovic etal. (1984) Science 224:497] and AIDS-associated retrovirus (ARV) [Levyet al. (1984) Science 225:840-842]. Applicants originally termed theseisolates “human T-cell lymphotropic retrovirus (hTLR)”. Subsequently,the name HIV has been given to these retroviruses by an internationalcommittee. Thus, HIV (and particularly HIV-1) shall be used herein as anequivalent to hTLR. Examples of HIV-1 were previously called LAV, ARVand HTLV-III. Among the identifying characteristics of HIV retrovirusesare (i) being an etiologic of AIDS, (ii) being cytopathic in vitro,(iii) having a tropism for CD4-bearing cells, and (iv) having elementstrans-activating the expression of viral genes acting at the LTR level.

New HIVs may be shown to be of the same class by being similar in theirmorphology, serology, reverse transcriptase optima, cytopathology, aminoacid sequence, and nucleotide sequence as known HIV strains. Coffin etal. (1986) Nature 321:10. Within different HIV-1 isolates, for example,the gag and pol proteins shows about 90-95% homology at the amino acidlevel, and the env precursor shows about 65-85% homology (most of thevariations being confined to certain “hypervariable” regions), with all23 env cysteines being conserved. Alizon et al. (1986) Cell 46:63-74.HIV-2, however, is a new class of the HIV family that is not a strain ofHIV-1 according to the recommended criteria of the internationaltaxonomy committee. See, e.g., Guyader et al. (1987) Nature 326:662-669.HIV-1 and HIV-2 show an overall approximate amino acid homology of about42%, with about 60% amino acid homology for the gag and pol proteins,and about 40% for the env precursor.

The nucleotide sequences of this invention may be the entire sequence ofthe retrovirus and/or the provirus or may be fragments thereof based onrestriction enzyme digestion of HIV (provirus and/or other dsDNAhomologous to retrovirus RNA), which fragments may be all or part of theLTR, gag, pol, env, and/or other open reading frames, such as Q (orsor), R, tat, and art (or trs) (sometimes referred to by the designation“orf” herein), untranslated regions intermediate coding regions, andfragments and combinations thereof. The minimum size single-strandedfragment will be at least 20 bases and usually at least 50 bases and maybe 100 bases or more, where the entire HIV is about 9.5 kb. The sequencemay be obtained as a fragment from the HIV or be synthesized.

The fragments can be used in a wide variety of ways, depending upontheir size, their natural function, the use for which they are desired,and the degree to which they can be manipulated to modify theirfunction. Thus, sequences of at least 20 bases, more usually at least 50bases, and usually not exceeding about 1000 bases, more usually notexceeding about 500 bases, may serve as probes for detection of thepresence of HIV in a host cell, including the genome, or in aphysiological fluid, such as blood, lymph, saliva, spinal fluid, or thelike. These sequences may include coding and/or non-coding sequences.The coding sequences may involve the gag, pol, env or other open readingframes, either in whole or in part. Where splicing occurs between, forexample, a region in the LTR sequence and a coding sequence in anotherregion, the joined DNA from the provirus, linked by in vitromanipulation, or from cDNA or cloned cDNA, may be employed.

It is found that HIV is highly polymorphic. Therefore, not only may DNAprepared from various isolates vary by one or more point mutations, buteven the passage of a single isolate may result in variation in theprogeny. Thus, where the nucleotide sequences are used for duplexformation, hybridization, or annealing, for example, for diagnosis ormonitoring of the presence of the virus in vivo or in vitro, completebase pairing will not be required. One or more mismatches arepermissible. To ensure that the presence of one or a few, usually notmore than three, mismatches still allows for stable duplexes under thepredetermined stringency of hybridizing or annealing conditions, probeswill normally be greater than 20 bases, preferably at least about 50bases or more.

The method of detection will involve duplex formation by annealing orhybridization of the oligonucleotide probe, either labeled or unlabeled,depending upon the nature of the detection system, with the DNA or RNAof a host suspected of harboring the provirus or virus. A physiologicalsample may include tissue, blood, saliva, serum, etc. Particularly,blood samples will be taken, more particularly blood samples containingperipheral mononuclear cells, which may be lysed and the DNA or RNAisolated in accordance with known techniques. Cells may be cultured toamplify virus in vitro, or treated to stimulate PBLs, thereby producingmore virus. Conveniently, the cells are treated with a detergent,nucleic acids are extracted with organic solvents and precipitated in anappropriately buffered medium, and the DNA or RNA isolated. Dependingupon the particular protocol, the DNA may be fragmented by mechanicalshearing or restriction endonuclease digestion.

The sample polynucleotide mixture obtained from the human host can bebound to a support or may be used in solution depending upon the natureof the protocol. The well-established Southern technique [(1975) J. Mol.Biol. 98:503] may be employed with denatured DNA, by binding thesingle-stranded fragments to a nitrocellulose filter. Alternatively, RNAcan be blotted on nitrocellulose following the procedure described byThomas, (1980) Proc. Natl. Acad. Sci. (USA) 77:5201. Desirably, thefragments will be electrophoresed prior to binding to a support, so asto be able to select for various sized fractions. Other techniques mayalso be used such as described in Meinkoth & Wahl, (1984) Anal. Biochem.138:267-284.

The oligonucleotide probe may be DNA or RNA, usually DNA. Theoligonucleotide sequence may be prepared synthetically or in vivo bycloning, where the complementary sequence may then be excised from thecloning vehicle or retained with the cloning vehicle. Various cloningvehicles are available, such as pBR322, M13, Charon 4A, or the like,desirably a single-stranded vehicle, such as M13.

As indicated, the oligonucleotide probe may be labeled or unlabeled. Awide variety of techniques exist for labeling DNA and RNA. Asillustrative of such techniques, is radiolabeling using nicktranslation, tailing with terminal deoxytransferase, or the like, wherethe bases which are employed carry radioactive ³²P. Alternatively,radioactive nucleotides can be employed where carbon, nitrogen or otherradioactive atoms may be part of the nucleoside structure. Other labelswhich may be used include fluorophores, enzymes, enzyme substrates,enzyme cofactors, enzyme inhibitors, or the like. Alternatively, insteadof having a label which provides for a detectable signal by itself or inconjunction with other reactive agents, ligands can be used to whichreceptors bind, where the receptors are labeled such as with theabove-indicated labels, which labels provide detectable signals bythemselves or in conjunction with other reagents. See, e.g., Leary etal. (1983) Proc. Natl. Acad. Sci. (USA) 80:4045-4049; Cosstick et al.(1984) Nucleic Acids Res. 12:1791-1810; PCT Pub. No. WO 83/02277.

The oligonucleotide probes are hybridized with the denatured human hostnucleic acid, substantially intact or fragmented, or fractions thereof,under conditions of predetermined stringency. The stringency will dependupon the size and composition of the probe, the degree of mismatching,the desired cross reactivity with other strains of the subject HIV, andthe like. Usually, an organic solvent such as formamide will be presentin from about 30 to 60 vol percent, more usually from about 40 to 50 volpercent, with salt concentration from 0.5 to 1 M. Temperatures willgenerally range from about 30° C. to 65° C., more usually from about 35°C. to 50° C. The times for duplex formation may be varied widely,although minimum times will usually be at least about one hour and notmore than about 72 hours, the time being selected in accordance with theamount of DNA or RNA available, the proportion of DNA or RNA as comparedto total DNA or RNA, or the like. Stringency may also be modified byionic strength and temperature. The hybridization and annealing can becarried out in two stages: a first stage in a hybridization medium; and,a second stage, involving washings at a higher stringency, by varyingeither or both temperature and ionic strength.

As understood in the art, the term “stringent hybridization conditions”as used herein refers to hybridization conditions which allow forclosely related nucleic acid sequences to duplex (e.g., greater thanabout 90% homology), but not unrelated sequences. The appropriateconditions can be established by routine procedures, such as runningSouthern hybridization at increasing stringency until only relatedspecies are resolved and the background and/or control hybridization hasdisappeared (i.e., selective hybridization).

The oligonucleotide probe may be obtained in a variety of ways. ViralRNA from HIV may be isolated from the supernatant of cells infected(e.g.; HIV-1 or HIV-2) in culture, and the high molecular weightmaterials precipitated and the DNA removed, for example, employingDNase. The residual RNA may then be divided into molecular weightfractions, where the fraction associated with the molecular weight ofthe retrovirus is isolated. This fraction will be from about 8 to 10 kbviral RNA. The viral RNA may be further purified by conventionaltechniques, such as electrophoresis, chromatography, or the like.

Nucleotide probes may be prepared employing reverse transcriptase usingprimers, e.g., random primers or specific primers. The cDNA may beprepared employing a radioactive label, e.g., ³²P, present with one ormore of the dNTPs. Reverse transcription will provide various sizedfragments depending on the primers, the efficiency of transcription, theintegrity of the RNA, and the like. The resulting cDNA sequences may becloned, separated and used for detection of the presence of a provirusin the human genome or for isolation of pure retroviral RNA.

Using specific primers of 10 to 20 bases, or more, HIV may be reversetranscribed and the resulting ss DNA used as a probe specific for theregion which hybridized to the primer. By employing one or moreradionucleotide-labeled bases, the probes will be radiolabeled toprovide a detectable signal. Alternatively, modified bases may beemployed which will be randomly incorporated into the probe and may beused to provide for a detectable signal. For example, biotin-modifiedbases may be employed. The resulting biotin-containing probe may then beused in conjunction with labeled avidin to provide for a detectablesignal upon hybridization and duplex formation.

Of particular interest is employing the region containing the gag or envgenes, where fragments may be employed to screen proviral DNA ininfected cells, to determine the identity of retroviruses associatedwith AIDS or LAS obtained from different human hosts. Probes providingfor the desired degree of cross-reactivity or absence ofcross-reactivity may then be prepared in a form, either labeled orunlabeled, useful for diagnostic assays employing hybridization andannealing.

The double-stranded DNA sequences, either isolated and cloned fromproviral DNA or cDNA or synthesized, may be used for expression ofpolypeptides which may be a precursor protein subject to furthermanipulation by cleavage, or a complete mature protein or fragmentthereof. The smallest sequence of interest, so as to encode an aminoacid sequence capable of specific binding, for example, to a receptor oran immunoglobulin, will be 21 bp, usually at least 45 bp, exclusive ofthe initiation codon. The sequence may code for any greater portion ofor the complete polypeptide, or may include flanking regions of aprecursor polypeptide, so as to include portions of sequences or entiresequences coding for two or more different mature polypeptides. Thesequence will usually be less than about 5 kbp, more usually less thanabout 3 kbp.

The sequences having open reading frames as numbered in FIG. 4 are thegenes beginning at nucleotide (nt) 838 to 2298 (gag); 2347 to 2825(small polypeptide between gag and pol regions); 2965 to 5103 (pol); and6236 to 8800 (env). It is to be understood that the above sequences maybe spliced to other sequences present in the retrovirus, so that the5′-end of the sequence may not code for the N-terminal amino acid of theexpression product. The splice site may be at the 5′-terminus of theopen reading frame or internal to the open reading frame. The initiationcodon for the protein may not be the first codon for methionine, but maybe the second or third methionine, so that employing the entire sequenceindicated above may result in an extended protein. However, for the gagand env genes there will be proteolytic processing in mammalian cells,which processing may include the removal of extra amino acids.

In isolating the different domains the provirus may be digested withrestriction endonucleases, the fragments electrophoresed and fragmentshaving the proper size and duplexing with a probe, when available, areisolated, cloned in a cloning vector, and excised from the vector. Thefragments may then be manipulated for expression. Superfluousnucleotides may be removed from one or both termini using Bal31digestion. By restriction mapping, convenient restriction sites may belocated external or internal to the coding region. Primer repair or invitro mutagenesis may be employed for defining a terminus, forinsertions, deletion, point or multiple mutations, or the like, wherecodons may be changed, either cryptic or changing the amino acid,restriction sites introduced or removed, or the like. Where the gene hasbeen truncated, the lost nucleotides may be replaced using an adaptor.Adaptors are particularly useful for joining coding regions to ensurethe proper reading frame.

The env domain of HIV can be obtained by digestion of the provirus withEcoRI and KpnI and purification of a 3300 base pair (bp) fragment, whichfragment contains about 400 bp of 5′ non-coding and about 200 bp of 3′non-coding region. Three different methionines coded for by the sequencein the 5′ end of the open reading frame may serve as translationalinitiation sites.

The open reading frame of the env gene of ARV-2 has a coding capacity of863 amino acids. Portions of the env gene coding for the polypeptidesshown in FIG. 5 were produced in S. cerevisiae using yeast expressionvectors. See FIG. 13. Env-2, encompassing amino acid residues 26 to 510,corresponds to the major portion of the mature envelope glycoprotein,gp120, that is external to viral and infected cell membranes. Env-1includes amino acid residues 26 to 276 and represents approximately theamino-terminal half of the gp120 polypeptide. Env-3, stretching betweenamino acid residues 529 to 855, corresponds to the portion of the envgene which encodes gp41, the viral glycoprotein that spans membranes andserves as an anchor for the envelope glycoprotein complex. Env-4, aminoacid residues 272 to 509, correspond to the carboxyl terminal half ofgp120. Env-5b, encompassing amino acid residues 557 to 677, correspondsto the region of gp41 stretching between the two hydrophobic domains.These various recombinant portions of the env domain are valuable indiagnostic assays for HIV infections, particularly env-2 and env-5b.

Digestion of proviral sequences with SacI and EcoRV provides a fragmentof about 2300 bp which contains the gag domain and a second small openreading frame towards the 3′ end of the gag region. The gag domain isabout 1500 bp and codes for a large precursor protein which is processedto yield proteins of about 25,000 (p25), 16,000 (p16) and 12,000 (p12)daltons. Digestion with SacI and BglII may also be used to obtainexclusively the gag domain with p12, p25 and partial p16 regions.

Digestion of the previous with KpnI and SstI provides a fragmentcontaining the portion of the pol domain that encodes p31. Native HIVreverse transcriptase (RT) is purified from virions in p66 and p51forms. Both of these forms have identical N-termini, apparentlydiffering at the C-termini. RT is encoded within a domain of the viralpol gene. The mature enzyme is derived by proteolytic processing from alarge precursor polypeptide whose cleavage is thought to be mediated bya viral protease. This protease, by analogy with other retroviruses,also cleaves the gag gene precursor. For direct expression of the RTdomain in yeast, the N- and C-termini of the mature protein wereestimated by drawing on homology comparisons with the amino acidsequences of pol gene products of other retroviruses. Precise amino acidchoices for termini were based on the target specificities of retroviralproteases, including the AIDS virus protease, from known gag subunitsequences. Accordingly, the Phe-Pro at positions 155 and 156 of theARV-2 pol open reading frame and the Val-Pro at positions 163 and 164were selected as likely N-termini. A likely C terminal processing sitewas estimated at the Val-Pro of positions 691 and 692. See FIG. 5.Recombinant RT is valuable in diagnostic assays for HIV infections.

The polypeptides which are expressed by the above DNA sequences may finduse in a variety of ways. The polypeptides or immunologically activefragments thereof, may find use as diagnostic reagents, being used inlabeled or unlabeled form or immobilized (i.e., bound to a solidsurface), as vaccines, in the production of monoclonal antibodies, e.g.,inhibiting antibodies, or the like.

The DNA sequences may be joined with other sequences, such as viruses,e.g., vaccinia virus or adenovirus, to be used for vaccination.Particularly, the DNA sequence of the viral antigen may be inserted intothe vaccinia virus at a site where it can be expressed, so as to providean antigen of HIV recognized as an immunogen by the host. The gag, pol,or env genes or fragments thereof that encode immunogens could be used.

Another alternative is to join the gag, env, or pol regions or portionsthereof to HBsAg gene or pre-S HBsAg gene or immunogenic portionsthereof, which portion is capable of forming particles in a unicellularmicroorganism host, e.g., yeast or mammalian cells. Thus, particles areformed which will present the HIV immunogen to the host in immunogenicform, when the host is vaccinated with assembled particles.

As vaccines, the various forms of the immunogen can be administered in avariety of ways, orally, parenterally, intravenously, intra-arterially,subcutaneously, intramuscularly, or the like. Usually, these will beprovided in a physiologically acceptable vehicle, generally distilledwater, phosphate-buffered saline, physiological saline, bufferscontaining SDS or EDTA, and the like. Various adjuvants may be included,such as aluminum hydroxide, MTP in saline and Tween 80, and the dosages,number of times of administration and manner ofadministration-determined empirically.

In order to obtain the HIV sequence (e.g., HIV-1 or HIV-2), virus can bepelleted from the supernatant of infected host cells. A 9 kb RNA speciesis purified by electrophoresis of the viral RNA in low-melting agarosegels, followed by phenol extraction. The purified RNA may then be usedas a template with random primers in a reverse transcriptase reaction.The resulting cDNA is then screened for hybridization to polyA+ RNA frominfected and uninfected cells, or to one of λ vectors containing HIV DNAdisclosed herein. For the polyA+ RNA, hybridization occurring frominfected, but not uninfected cells, is related to HIV.

Genomic DNA from infected cells can be digested with restriction enzymesand used to prepare a bacteriophage library. Based upon restrictionanalysis of the previously obtained fragments of the retrovirus, theviral genome can be partially digested with EcoRI and 9 kb-15 kb DNAfragments isolated and employed to prepare the library. The resultingrecombinant phage may be screened using a double-lift screening methodemploying the viral cDNA probe, followed by further purification, e.g.,plaque-purification and propagation in large liquid cultures. From thelibrary, the complete sequence of the virus can be obtained and detectedwith the previously described probe.

HIV DNA (either provirus or cDNA) may be cloned in any convenientvector. Constructs can be prepared, either circular or linear, where theHIV DNA, either the entire HIV or fragments thereof, may be ligated to areplication system functional in a microorganism host, eitherprokaryotic or eukaryotic cells (mammalian, yeast, arthropod, plant).Micro-organism hosts include E. coli, B. subtilis, P. aerugenosa, S.cerevisiae, N. crassa, etc. Replication systems may be derived fromColE1, 2 mμ plasmid, λ, SV40, bovine papilloma virus, or the like, thatis, both plasmids and viruses. Besides the replication system and theHIV DNA, the construct will usually also include one or more markers,which allow for selection of transformed or transfected hosts. Markersmay include biocide resistance, e.g., resistance to antibiotics, heavymetals, etc., complementation in an auxotrophic host to provideprototrophy, and the like.

To produce recombinant polypeptides, expression vectors will beemployed. For expression in microorganisms, the expression vector maydiffer from the cloning vector in having transcriptional andtranslational initiation and termination regulatory signal sequences andmay or may not include a replication system which is functional in theexpression host. The coding sequence is inserted between the initiationand termination regulatory signals so as to be under their regulatorycontrol. Expression vectors may also include the use of regulablepromoters, e.g., temperature-sensitive or inducible by chemicals, orgenes which will allow for integration and amplification of the vectorand HIV DNA such as tk, dhfr, metallothionein, or the like.

The expression vector is introduced into an appropriate host where theregulatory signals are functional in such host. The expression host isgrown in an appropriate nutrient medium, whereby the desired polypeptideis produced and isolated from cells or from the medium when thepolypeptide is secreted.

Where a host is employed in which the HIV transcriptional andtranslational regulatory signals are functional, then the HIV DNAsequence may be manipulated to provide for expression of the desiredpolypeptide in proper juxtaposition to the regulatory signals.

The polypeptide products can be obtained in substantially pure form,particularly free of debris from human cells, which debris may includesuch contaminants as proteins, polysaccharides, lipids, nucleic acids,viruses, bacteria, fungi, etc., and combinations thereof. Generally, thepolypeptide products will have less than about 10-15 weight percent,preferably less than about 5 weight percent, of contaminating materialsfrom the expression host. Depending upon whether the desired polypeptideis produced in the cytoplasm or secreted, the manner of isolation willvary. Where the product is in the cytoplasm, the cells are harvested,lysed, the product extracted and purified, using solvent extraction,chromatography, gel exclusion, electrophoresis, or the like. Wheresecreted, the desired product will be extracted from the nutrient mediumand purified in accordance with the methods described above.

In many cases it will be desirable to express the recombinant HIVpolypeptide as a fusion protein. This is particularly true withpolypeptides such as p31pol and the transmembrane region of gp41env(env-5), to obtain improved levels of expression. The fusion proteinsapproach allows the addition of a signal sequence to the HIV polypeptideso that the product is secreted by the expression host. Generally, theDNA sequence for the HIV polypeptide is in the C-terminal portion of thefused gene, the heterologous sequence making up the N-terminal. Thechoice of the appropriate heterologous sequence for fusion to the HIVsequence is a matter of choice within the skill of the art. Preferredheterologous sequences include the N-termini of β-galactosidase andhuman superoxide dismutase. It is usually preferable that theheterologous sequence be non-immunogenic to humans. In one embodiment,however, two HIV sequences from different immunogenic domains of thevirus, such as gag and env, are fused together. This produces a singlefusion protein with the immunogenic potential of the two parentpolypeptides.

The expression products of the env, gag, and pol genes and immunogenicfragments thereof having immunogenic sites may be used for screeningantisera from patients' blood to determine whether antibodies arepresent which bind to HIV antigens. One or more of the antigens may beused in the assay. Preferred modes of the assay employ a combination ofgag and env antigens or pol and env antigens. A combination of p25gag,p16gag, or p31pol and env antigens is particularly preferred. A widevariety of assay techniques can be employed, involving labeled orunlabeled antigens or immobilized antigens. The label may befluorescers, radionuclides, enzymes, chemiluminescers, magneticparticles, enzyme substrates, cofactors or inhibitors, ligands, or thelike.

A particularly convenient technique is to bind the antigen to a supportthat will bind proteins, such as the surface of an assay tube, a well ofan assay plate, or a strip of material like nitrocellulose or nylon, andthen contact the sample with the immobilized antigen. After washing thesupport to remove non-specifically bound antisera, labeled antibodies tohuman Ig are added and specifically bound label determined.

ELISA and “dot-blot” assays are particularly useful for screening bloodor serum samples for anti-HIV antibodies. The ELISA assay usesmicrotiter trays having wells that have been coated with the antigenicHIV polypeptides(s). The wells are also typically post-coated with anonantigenic protein to avoid nonspecific binding of antibodies in thesample to the well surface. The sample is deposited in the wells andincubated therein for a suitable period under conditions favorable toantigen-antibody binding.

Anti-HIV antibodies present in the sample will bind to the antigen(s) onthe well wall. The sample is then removed and the wells are washed toremove any residual, unbound sample. A reagent containing enzyme-labeledantibodies to human immunoglobulin is then deposited in the wells andincubated therein to permit binding between the labeled anti-human Igantibodies and HIV antigen-human antibody complexes bound to the wellwall. Upon completion of the incubation, the reagent is removed and thewells washed to remove unbound labeled reagent. A substrate reagent isthen added to the wells and incubated therein. Enzymatic activity on thesubstrate is determined visually or spectrophotometrically and is anindication of the presence and amount of anti-HIV antibody-containingimmune complex bound to the well surface.

The “dot-blot” procedure involves using HIV antigen(s) immobilized on apiece or strip of bibulous support material, such as nitrocellulosefilter paper or nylon membrane, rather than antigen-coated microtitertrays. The support will also be treated subsequently with a nonantigenicprotein to eliminate nonspecific binding of antibody to the support. Theantigen-carrying support is contacted with (e.g., dipped into) thesample and allowed to incubate therein. Again, any anti-HIV antibodiesin the sample will bind to the antigen(s) immobilized on the support.After a suitable incubation period the support is withdrawn from thesample and washed in buffer to remove any unbound sample from the paper.The support is then incubated with the enzyme-labeled antibody to humanIg reagent for a suitable incubation period. Following treatment withthe labeled reagent the support is washed in buffer, followed byincubation in the substrate solution. Enzymatic activity, indicating thepresence of anti-HIV antibody-containing complexes on the support,causes color changes on the support which may be detected optically.

Either of these techniques may be modified to employ labels other thanenzymes, or to detect non-human anti-HIV antibodies (e.g., primate). Thereading or detection phases will be altered accordingly.

The antigenic HIV polypeptide may also be used as immunogens bythemselves or joined to other antigens for the production of antisera ormonoclonal antibodies which may be used for therapy or diagnosis. Whenused as immunogens, the HIV polypeptides can be prepared as vaccinecompositions, as is known in the art. The immunoglobulins may be fromany mammalian source, e.g., rodent, such as rat or mouse, primate, suchas baboon, monkey or human, or the like. For diagnosis, the antibodiescan be used in conventional ways to detect HIV in a clinical sample.

EXAMPLES

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the claims in any way. Theexamples are organized as follows:

1. Isolation, Cloning and Characterization of HIV

-   -   1.1. Purification and preparation of viral RNA        -   1.2. Synthesis of labeled viral probe        -   1.3. Detection of HIV RNA and DNA in mammalian cells        -   1.4. Cloning of proviral HIV DNA        -   1.5. Polymorphism of HIV        -   1.6. Sequencing of proviral DNA        -   1.7. Amino acid sequences of native HIV proteins            2. Expression of HIV Polypeptides in Mammalian Cells    -   2.1. Expression of env peptide        -   2.1.1. Mammalian expression vector pSV-7c        -   2.1.2. Transformation of cells with env sequences        -   2.1.3. Detecting expression via immunofluorescence    -   2.2 Expression of gp160env        -   2.2.1. Mammalian expression vector pSV7d        -   2.2.2. Expression of tPA/gp160    -   2.3 Expression of gp120env        -   2.3.1. Expression of tPA/gp120 in pSV7d        -   2.3.2. Expression of gp120 using CMV IE-1 promoter    -   2.4 Expression of gag peptides    -   2.5 Expression of gag-env fusion protein        3. Expression of HIV Polypeptides in Bacteria    -   3.1. Expression of p25gag    -   3.1.1. Host-Vector System    -   3.1.2. Construction of pGAG25-10    -   3.1.3. Transformation and expression of gag peptide    -   3.1.4. Fermentation process        -   3.1.4.1. Preparation of master seed stock        -   3.1.4.2. Fermenter inoculum        -   3.1.4.3. Fermentation and harvest    -   3.1.5. Purification of p25gag    -   3.1.6. Characterization of recombinant p25gag        -   3.1.6.1. Protein gel electrophoresis        -   3.1.6.2. Western analysis        -   3.1.6.3. ELISA comparison of recombinant and native p25gag    -   3.2. Expression of p16gag    -   3.3. Expression of fusion protein p41gag    -   3.4. Expression of p31pol        -   3.4.1. Host-Vector System        -   3.4.2. Construction of pTP31.2            -   3.4.2.1. Construction of M13 template 01100484            -   3.4.2.2. In vitro mutagenesis of 01100484            -   3.4.2.3. Isolation of DNA fragments containing p31            -   3.4.2.4. Cloning of p31 into plot 7            -   3.4.2.5. Construction of pTP31        -   3.4.3. Screening of transformants        -   3.4.4. Characterization of recombinant protein    -   3.5. Expression of SOD-p31 fusion protein    -   3.6. Expression of SOD-env5b fusion protein        -   3.6.1. Host-Vector System        -   3.6.2. Construction of pSOD/env5b        -   3.6.3. Expression of SOD/env5b fusion protein        -   3.6.4. Protein Purification    -   3.7. Expression of β-gal-env fusion proteins        -   3.7.1. Host-Vector System        -   3.7.2. Construction of pII-3        -   3.7.3. Expression and characterization of fusion protein            4. Expression of HIV Polypeptides in Yeast    -   4.1. Expression of envH peptide        -   4.1.1. Host-Vector System        -   4.1.2. Construction of pDPC303        -   4.1.3. Transformation and expression        -   4.1.4. Purification of envH protein            -   4.1.4.1. Cell breakage            -   4.1.4.2. SDS extraction of insoluble material            -   4.1.4.3. Selective precipitation and gel filtration        -   4.1.5. Characterization of recombinant envH    -   4.2. Expression of env subregion polypeptides        -   4.2.1. Env-1            -   4.2.1.1. GAP promoter        -   4.2.2. Env-2            -   4.2.2.1. Construction of pAB24-GAP-env2            -   4.2.2.2. Transformation and expression            -   4.2.2.3. Purification of env-2 protein            -   4.2.2.4. Immunogenicity        -   4.2.3. Env-3            -   4.2.3.1. GAP promoter            -   4.2.3.2. ADH-2/GAPDH promoter        -   4.2.4. Env-4            -   4.2.4.1. pBS24/SF2env4/GAP            -   4.2.4.2. pBS24            -   4.2.4.3. pBS24/SOD-SF2env4        -   4.2.5. ySOD/env-5b fusion protein        -   4.2.6. Env 4-5            -   4.2.6.1. Construction of pBS24.1/SOD-SF2env 4-5            -   4.2.6.2. Transformation and expression            -   4.2.6.3. Protein purification    -   4.3. p31pol        -   4.3.1. GAP/ADH2 promoter        -   4.3.2. GAP promoter        -   4.3.3. SOD-p31 fusion protein            -   4.3.3.1. pCl/1-pSP31-GAP-ADH2            -   4.3.3.2. Transformation and expression            -   4.3.3.3. Purification and characterization    -   4.4 Reverse transcriptase (RT)        -   4.4.1. pAB24/RT4 expression vector        -   4.4.2. Transformation and expression        -   4.4.3. Purification        -   4.4.4. Electrophoresis and immunoblotting        -   4.4.5. RT activity assay    -   4.5. p25gag        -   4.5.1. Host-Vector System        -   4.5.2. Saccharomyces cerevisiae AB110        -   4.5.3. pCl/1-p25-ADH-GAP        -   4.5.4. Transformation and expression        -   4.5.5. Protein Purification    -   4.6. p53gag        -   4.6.1. Construction of pCl/1-GAP-p53        -   4.6.2. Transformation and expression        -   4.6.3. Protein purification            5. Immunoassays for Anti-HIV Abs Using Recombinant HIV            Polypeptides    -   5.1. ELISA-A        -   5.1.1. Assay Protocol        -   5.1.2. Results    -   5.2. ELISA-B        -   5.2.1. Assay Protocol        -   5.2.2. Results    -   5.3. Dot Blot Assay    -   5.4. Immunoblot Strip ELISA Assay        6. Serology Studies with Recombinant HIV Polypeptides    -   6.1. Immunoblot with Env Polypeptides    -   6.2. ELISA with Env Polypeptides    -   6.3. ELISA with gag Polypeptides    -   6.4. Western and ELISA with Pol Polypeptides        7. HIV Immunization    -   7.1. Immunization of mice    -   7.2. Immunization of guinea pigs        7. Deposits of Biological Materials        1. Isolation, Cloning and Characterization of HIV

The DNA and RNA sequences of HIV are provided, as well as fragmentsthereof, which find extensive use in the detection of the presence ofHIV, for the expression of protein specific for HIV and the use of suchproteins for the production of monoclonal antibodies for in vitro and invivo use, in diagnostics, therapy, or the like. In addition, due to theobserved polymorphism of HIV, probes are indicated which can be used todetect the presence of HIV or a particular polymorphism thereof. Theprobes are at least about 20 bases and will usually not be more thanabout 500 bases and may be in the gag, env or LTR region. Furthermore, astrategy is provided for analyzing the various polymorphisms, usingrestriction enzyme analysis, whereby different isolates can be relatedin accordance with different families.

1.1 Purification and Preparation of Viral RNA

HUT-78 cells infected with ARV-2 (ATCC Accession No. CRL 8597, depositedon Aug. 7, 1984) were obtained from Dr. Jay Levy, University ofCalifornia, San Francisco. Cultures were grown for two weeks in RPMImedium with 10% fetal calf serum. Cells were removed by low-speedcentrifugation (1,000×g for 10 min), and the resulting supernatantscentrifuged at 2 Krpm for 1 h at 4° C. using a SW-28 rotor. The pellet,containing the virus, was resuspended in 10 mM Tris-HCl, pH 7.5 on ice.The resuspended pellet was treated with 10 μg of DNase(Boehringer-Mannhein) and was layered onto a linear sucrose gradient(15-50% in 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 20 mM NaCl). The gradientwas spun at 34 Krpm for 4 h at 4° C., in SW-41 rotor. Five 2.5 mlfractions were collected and an aliquot of each was electrophoresed in a1% agarose, 5 mM methyl mercury hydroxide gel [Bailey et al. (1976)Anal. Biochem. 70:75-85] to determine which contained the 9 kb viralRNA. The fraction containing the viral RNA (identified by gel analysis)was diluted to 10 ml in 10 mM Tris-HCl, pH 7.5, 1 mM EDTA and wascentrifuged at 34 Krpm for 2 h at 4° C. The pellet was resuspended in 20mM Tris-HCl, pH 7.6, 10 mM EDTA, 0.1% SDS, and 200 μg/ml proteinase K.Incubation was carried out for 15 min at room temperature. The mixturewas extracted with phenol and the aqueous phase was made 400 mM NaCl andprecipitated with ethanol. The pellet was resuspended in water andstored at −70° C.

To purify the viral RNA from the nucleic acid pellet obtained asdescribed above, a sample was electrophoresed in a low-melting 1%agarose gel containing 5 mM Methyl mercury hydroxide. Afterelectrophoresis, the gel was stained with 0.1% ethidium bromide andnucleic acid bands were visualized under UV light. The regioncorresponding to 9 kb was cut from the gel and the agarose was melted at70° C. for 2 to 3 min in three volumes of 0.3 M NaCl, 10 mM Tris, pH7.5, 1 mM EDTA. The mixture was extracted with an equal volume ofphenol. The aqueous phase was reextracted with phenol and wasprecipitated with ethanol. The pellet was washed with cold 95% ethanol,air dried, resuspended in water and stored at −70° C. until use. Onehundred ml of culture medium yielded 0.5 to 1 μg of purified RNA.

1.2. Synthesis of Labeled Viral Probe

A ³²P-labeled cDNA was made to the gel purified viral RNA using randomprimers (calf thymus primers) prepared as described in Maniatis et al.,Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratories1982). The reaction mixture contained 2 μl of 0.5 M MgCl₂; 5 μl of 0.1 Mdithiothreitol; 2.5 μl each of 10 mM dATP, 10 mM dGTP and 10 mM dTTP;2.5 μl calf thymus primer (100A₂₆₀/ml); 0.5 μg viral RNA; 5 μl ofactinomycin D (200 μg/ml); 10 μl of ³²P-dCTP (>3000 Ci/mmole, 1 mCi/ml)and 1 μl of AMV reverse transcriptase (17 units/μl) in a 50 μl reactionvolume. The reaction was incubated for 1 h at 37° C. The probe waspurified away from free nucleotides by gel filtration using a SephadexG50 column. The void volume was pooled, NaCl was added to a finalconcentration of 400 mM and carrier single-stranded DNA to 100 μg/ml,and the cDNA was precipitated with ethanol. The pellet was resuspendedin water and incorporated ³²P counts were determined.

1.3. Detection of HIV RNA and DNA in Mammalian Cells

PolyA+ RNA was prepared from HUT-78 cells infected with ARV-2, ARV-3 orARV-4 (three different isolates from three different AIDS patients) andfrom uninfected HUT-78 cells. The polyA+ RNA was electrophoresed on 1%agarose gels containing 5 mM methyl mercury hydroxide (Bailey et al.supra), was transferred to nitrocellulose filters, and hybridized withthe homologous probe prepared as described in Section 1.2.Hybridizations were carried out in 50% formamide, 3×SSC at 42° C. Washeswere at 50° C. in 0.2×SSC. A 9 kbp band was present in all three samplesof infected HUT-78 cells. This band was absent in polyA+ from uninfectedcells.

High molecular weight DNA (chromosomal) was prepared from cultures ofHUT-78 cells infected with ARV-2 and from non-infected HUT-78 cellsfollowing the procedure of Luciw et al. (1984) Molec. & Cell Biol.4:1260-1269. The DNA was digested with restriction enzyme(s),electrophoresed in 1% agarose gels and blotted onto nitrocellulosefollowing the procedure described by Southern, (1975), supra. Blots werehybridized with the ³²P-labeled probe (10⁶ cpm/blot) in a mixturecontaining 50% formamide, 3×SSC, 10 mM Hepes, pH 7.0, 100 μg/mldenatured carrier DNA, 100 μg/ml yeast RNA and 1× Denhardt's for 36 h at42° C. Filters were washed once at room temperature in 2×SSC and twiceat 42° C. in 0.2×SSC, 0.1% SDS. Filters were air dried and exposed toX-Omat film using an intensifying screen.

The homologous ³²P-probe to ARV-2 hybridized specifically to two bandsin the DNA from infected cells restricted with SacI. These bands wereabsent when DNA of non-infected cells was used, indicating that theprobe is hybridizing specifically to infected cells presumably to theprovirus integrated in the chromosomal DNA. The molecular weight of thebands is approximately 5 kb and 3 kb.

In order to determine if different enzymes would cut the proviralsequence, several other restriction digestions of the cell DNA werecarried out using EcoRI, SphI or KpnI or double digestions using two ofthem. Southern results show specific bands hybridizing when DNA ofinfected cells is used. FIG. 1 shows a schematic map of the positions ofrestriction enzyme sites in the proviral sequence, and indicatesfragment sites.

1.4. Cloning of Proviral HIV DNA

High molecular weight cell DNA from infected HUT-78 cells was preparedfollowing the procedure of Luciw et al., supra. The DNA was digestedwith EcoRI, which cuts once in the provirus, centrifuged in a sucrosegradient and fractions corresponding to 8-15 kb were pooled, dialyzedand concentrated by ethanol precipitation. The bacteriophage λderivative cloning vector, EMBL-4 [Karn et al. (1983) Methods Enzymol.101:3-19] was digested to completion with a mixture of EcoRI, BamHI andSalI restriction enzymes and the DNA then deproteinized byphenol-chloroform extraction, precipitated with cold ethanol andresuspended in ligation buffer. The EMBL-4 phage DNA and EcoRI digest ofcellular DNA were mixed and ligated and the resultant recombinant phagegenomes packaged in vitro. After phage infection of λ-sensitive E. coli(DP50supF), about 500,000 phage plaques were transferred ontonitrocellulose filters, DNA was fixed and the filters were screened witha homologous ³²P-probe prepared as described in Section 1.2. Elevenrecombinant phage out of 500,000 phage annealed in the initialdouble-lift screening method (Maniatis et al., supra) to viral cDNAprobe, and these were further plaque-purified and propagated in largeliquid cultures for preparation of recombinant DNA. Plaque-purifiedphage containing ARV DNA were propagated in liquid culture in E. coliDP50supF; phage particles were harvested and banded in CsCl gradientsand recombinant phage DNA was prepared by phenol extraction followed byethanol precipitation (Maniatis et al., supra). One microgram ofpurified phage DNA was digested with restriction enzymes,electrophoresed on 1% agarose gels, and visualized with ethidium bromideunder ultraviolet light. The DNA from these gels was transferred tonitrocellulose and annealed with viral cDNA probe.

Described below is the analysis of the 11 recombinant phage DNAmolecules utilizing restriction enzymes and viral cDNA probe. Twoexamples were selected for detailed description: λ-9B contained aninsertion of full-length proviral DNA along with flanking cellsequences, and λ-7A harbored a full-length viral genome permuted withrespect to the EcoRI site. Digestion of λ-9B DNA with SacI yielded viralDNA fragments of 3.8 kb and 5.7 kb (FIG. 2). EcoRI digestion of λ-9Bproduced virus containing DNA species at 6.4 kb and 8.0 kb; a doubledigest of SacI and EcoRI gave viral DNA fragments at 3.8 kb and 5.4 kb(FIG. 2). This pattern is consistent with that of a provirus linked tocell DNA. The patterns of the digestion with KpnI and with a mixture ofKpnI and EcoRI support this conclusion for λ-9B. In particular,digestion of λ-9B DNA with KpnI showed a 5.2 kb species that representsthe internal fragment seen in proviral DNA (FIGS. 1 and 2).Bacteriophage λARV-2(9B) was deposited at the ATCC on 25 Jan. 1985 andgiven Accession No. 40158. A second recombinant phage, λ-10C, alsocontained a full-length proviral DNA insertion.

When the DNA of λ-7A phage was treated with SacI, two viral DNAfragments were observed at 3.8 kb and 6.6 kb (FIG. 3). EcoRI digestionof λ-7A DNA produced a 9.4 kb viral species, and a digestion with amixture of EcoRI and SacI yielded two viral DNA fragments at 3.8 kb and5.4 kb (FIG. 3). Thus, λ-7A should represent a recombinant phage clonecontaining a full-length linear HIV genome that is permuted with respectto the EcoRI site. Analyses with KpnI support this model. Afterdigestion with KpnI a DNA fragment at 4.2 kb was observed as well asother DNA species. See FIG. 3. The data indicated that the 4.2 kb DNAfragment represents the circle junction and the others represent HIV DNAlinked to cell DNA and bacteriophage vector DNA. The double digestionwith KpnI and EcoRI left the 4.2 kb DNA intact and produced twofragments at 1.6 kb and 3.6 kb (FIG. 3); these three DNA species addedup to 9.4 kb and constituted the HIV genome predicted from a permutedconfiguration.

In addition to the two types of recombinant phage containing HIV DNAdescribed above, phage was obtained that (1) possessed the left half ofthe viral genome from the EcoRI site in viral DNA extending intoflanking cell DNA [λARV-2(8A)] and (2) phage that had the right halt ofthe viral genome [λARV-2(7D)] from the EcoRI site in viral DNA extendinginto flanking cell. DNA. The four types of recombinant phage DNAstructures are predicted from the analysis of viral DNA observed ininfected cells. Maps of restriction enzyme sites are shown in FIG. 1-3.The data for these maps were compiled from studies of proviral DNA ininfected cells, from characterization of recombinant phage DNA, and frompreliminary DNA sequence information. Bacteriophages λARV-2(7D) (right)and λARV-2(8A) (left) were deposited at the ATCC on Oct. 26, 1984 andgiven Accession Nos. 40143 and 40144, respectively.

To validate the cloned HIV DNA, a radioactive probe was prepared fromtwo regions of the cloned HIV genome that represent about 70% of thegenome and this probe was used to detect HIV DNA in restriction enzymedigests of DNA from infected cells. Whole-cell DNA was prepared fromHUT-78 cell cultures infected with HIV-1 strains ARV-2, ARV-3 and ARV-4,and analyzed by digestion with restriction enzymes as described inSection 1.3. A probe to cloned ARV-2 DNA was prepared as follows: DNAfrom recombinant phage λ-7A (FIG. 3) was digested with SacI or with amixture of SacI and KpnI and digestion products were eiectrophoresed in1% agarose gels (low-melting agarose); the 3.8 kb DNA fragment from thedigestion with SacI and the 3.1 kb DNA fragment from the digestion withSacI and KpnI (FIG. 3) were eluted, pooled, denatured by boiling inwater for 2 min, and used as templates with random DNA primers (calfthymus) with reverse transcriptase.

DNA from HUT-78 cells infected with ARV-2, ARV-3 or ARV-4 were digestedwith SacI, PstI or HindIII. Digested DNA was electrophoresed on 1%agarose gels, blotted onto nitrocellulose filters and annealed withprobe to cloned ARV-2 DNA or to homologous cDNA probe prepared asdescribed in Section 1.2. Results show that SacI, PstI, and HindIIIfragments detected by both probes are identical in all isolates.

1.5. Polymorphism of HIV

To measure the relatedness of independent HIV isolates, restrictionenzyme digests of DNA from HUT-78 cells infected with ARV-3 and ARV-4were analyzed with the probe made from cloned ARV-2 DNA. The SacI digestof ARV-3 DNA was similar to that of ARV-2 whereas the HindIII digestsdisplayed different patterns. The SacI digest and the PstI digest ofARV-4 DNA differed from the corresponding digests of ARV-2 DNA. Theintensity of the annealing signals obtained with ARV-3 and ARV-4 sampleswas much lower (about 10-fold less) than that for ARV-2 DNA, probably asa result of the fact that fewer cells were infected in the ARV-3 andARV-4 cultures. The viral-specific DNA fragments produced by SacItreatment of ARV-3 and ARV-4 DNA totaled 9.0-9.5 kbp, a value similar tothat of ARV-2 and in consonance with the RNA genome sizes.

1.6. Sequencing of Proviral DNA

Fragments or subfragments of ARV-2 DNA were prepared from therecombinant phages and cloned into M13 according to conventionalprocedures (Maniatis et al., supra). Sequencing was performed accordingto Sanger et al. [(1977) Proc. Natl. Acad. Sci. USA 74:5463], using theuniversal M13 primer or chemically synthesized primers complementary toARV-2 sequence. The sequence is shown in FIG. 4. Also indicated in thisfigure are the restriction sites present in the DNA and the open readingframes encoded by the sequence.

The sequence of the HIV-1 DNA from λ phage 9B was sequenced in a similarmanner. This sequence is shown in FIG. 5. There are several differencesbetween the sequences shown in FIGS. 4 and 5 which reflect thepolymorphism in HIV and the fact that the sequence of FIG. 4 was derivedas a composite from sequence data on several HIV isolates, whereas thesequence of FIG. 5 is from a single isolate. The main differenceaffecting polypeptide sequence is that the small open reading framebetween the gag and pol genes in FIG. 4 is not independent, but is partof the pol gene in FIG. 5. This merger of these reading frames was theresult of three base changes. The region of fusion and sequence changeoccurs roughly between nucleotides 2853 and 2941 in FIG. 5.

Furthermore, open reading frames in FIG. 4 were translated into aminoacids beginning with the first methionine in the open reading frame,whereas in FIG. 5 translation into amino acids was begun immediatelywith the codon following the stop codon.

1.7. Amino Acid Sequences of Native HIV Proteins

ARV-2 was prepared and purified as described in Section 1.1. The viralproteins were electrophoresed on an acrylamide gel, and the bandcorresponding to a 24,000 dalton or 16,000 dalton protein was excisedfrom the gel and used for sequencing. Micro-sequence analysis wasperformed using Applied Biosystems model 470A protein sequencer similarto that described by Hewick et al. (1981) J. Biol. Chem. 256:7990-7997.Phenylthiohydantoin amino acids were identified by HPLC using a BeckmanUltrasphere ODS column and a trifluoroacetic acid-acetonitrile buffersystem as reported by Hawke et al. (1982) Anal. Biochem. 120:308-311.Table 1 shows the first 20 amino acids from the amino terminusdetermined for p25-gag protein and Table 2 shows the first 30 aminoacids for p16-gag protein.

TABLE 1 Amino-terminal sequence of p25gag Position Amino acid 1 Pro 2Ile 3 Val 4 Gln 5 Asn 6 Leu 7 Gln 8 Gly 9 Gln 10 Met 11 Val 12 (His) 13Gln 14 Ala 15 Ile 16 (Ser) 17 Pro 18 (Arg, Lys) 19 Thr 20 (Leu)

TABLE 2 Amino-terminal sequence of p16gag Position Amino acid 1 (Met) 2Gln 3 Arg 4 Gly 5 Asn 6 Phe 7 Arg 8 Asn 9 Gln 10 Arg 11 Lys 12 Thr 13Val 14 Lys 15 ---(Cys) 16 Phe 17 Asn 18 ---(Cys) 19 Gly 20 Lys 21 Glu 22Gly 23 (His) 24 Ile 25 Ala 26 (Lys) 27 Asn 28 (Gly) 29 (Arg) 30 (Ala,Leu)

The amino acid sequence of Table 1 is predicted by nucleotides 1195 to1255, and from Table 2 by nucleotides 1930 to 2120, from the ARV-2 DNAsequence (indicated by bars in FIG. 4). Therefore, these results confirmthat the indicated gag open reading frame is in fact being translatedand identifies the N-termini of p25 and p16.

2. Expression of HIV Polypeptides in Mammalian Cells

2.1. Expression of Env Peptide

2.1.1. Mammalian Expression Vector pSV-7c

Plasmid pSV-7c contains the SV40 early promoter, origin of replicationand polyA processing signal, and was constructed as described below.

A 400 bp BamHI-HindIII fragment containing the SV40 origin ofreplication and early promoter was obtained by digestion of plasmidpSVgt1 (P. Berg, Stanford University, Palo Alto, Calif.) andpurification through gel electrophoresis. A second 240 bp SV40 fragmentcontaining SV40 polyA addition sites was obtained by digestion ofpSV2/dhfr [Subramani et al. (1981) J. Mol. Cell. Biol. 1:854-864] withBclI and BamHI, and gel purification. Both fragments were fused togetherthrough a linker which provided for a HindIII overhang in the 5′-end, aBclI overhang in the 3′-end, three general restriction sites and threesuccessive stop codons in all three reading frames. The sequence of thepolylinker was the following:

                   Stop Codons                       1   2   35′-AGCTAGATCTCCCGGGTCTAGATAAGTAAT-3′       TCTAGAGGGCCCAGATCTATTCATTACTAG HindIII  BglIISmaI  XbaI       Bc1I overhangThe about 670 bp fragment, containing SV40 origin of replication, SV40early promoter, polylinker with stop codons and SV40 polyadenylationsite, was cloned into the BamHI site of pML [Lusky & Botchan, (1984)Cell 36:391], a pBR322 derivative with an about 2.5 kbp deletion, toyield pSV-6. To eliminate the EcoRI and EcoRV sites present in pMLsequences of pSV-6, this plasmid was digested with EcoRI and EcoRV,treated with Bal31 nuclease to remove about 200 bp per end, religatedand cloned to yield pSV-7a. The Bal31 resection also eliminated oneBamHI restriction site flanking the SV40 region approximately 200 bpaway from the EcoRV site. To further eliminate the other BamHI siteflanking the SV40 region, pSV-7a was digested with NruI, which cuts inthe SV40 sequence, and PvuII, which cuts in the pML sequence upstreamfrom the origin of replication. The plasmid was recircularized by bluntend ligation and cloned to yield pSV-7b. To increase the number ofcloning sites of pSV-7b, a new polylinker was cloned into the plasmid toreplace the previous one but the stop codons in the three frames werestill retained. For this purpose, pSV-7b was digested with StuI andXbaI, and a linker of the following sequence was ligated to the vectorto yield pSV-7c.

    BglII EcoRI  SmaI   KpnI XbaI 5′-AGATCTCGAATTCCCCGGGGGTACCT-3′   TCTAGAGCTTAAGGGGCCCCCATGGAGATC

2.1.2. Transformation of Cells with Env Sequences

The HIV DNA coding for the env region was excised from a recombinantphage λARV-2(7D) [λ7D] which contains the right half of the provirus bydigestion with KpnI and EcoRI and gel purification of about 3300 bpfragment. This fragment was cloned into plasmid pSV-7c as shown in FIG.6. The recombinant vector. (pSV7c/env) was used to transfect Cos cells[Mellon et al. (1981) Cell 27:279] following the procedure of van der Eb& Graham [(1980) Methods in Enzymology 65:826-839] as modified by Parkerand Stark [(1979) J. Virol. 31:360369], in 60 mm plastic dishes (5×10⁵cells per dish). Thirty-six hours later the cells were transferred toglass microscope slides and cultured for another 24 h (5×10⁴ cells perchamber in a 4-chamber Titer-tek slide). The cell sheet was rinsed inPBS and fixed by dipping in cold acetone for 5 sec.

2.1.3. Detecting Expression Via Immunoflorescence

The fixed-cell monolayers in each chamber were incubated with AIDSpatient sera (Reference serum EW511 obtained from Dr. Paul Feorino,Center for Disease Control) and with control normal human sera. Bothsera were diluted 1/100 in PBS and 50 microliters were applied perchamber, incubated for 1 h at 37° C., and rinsed by dipping in PBS.Fluoresceinated goat anti-human antisera (Cappel Labs) was diluted 1/100in PBS and 50 microliters were applied to each chamber and incubated for30 min at 37° C. After washing in PBS, 75% glycerol in PBS was added tothe cell sheets, a coverslip was placed on top, and the cells wereobserved in a fluorescence microscope. About five cells in 100 showedbright immunofluorescence with the AIDS sera. No cells stained withcontrol normal human sera. Cells transfected with a control plasmidcontaining only SV40 vector sequences did not show anyimmunofluorescence with any antisera. Thus, the EcoRI-KpnI ARV-2 DNAfragment encodes a gene that can be expressed in mammalian cells and theexpression product detected specifically by AIDS patient sera.

2.2. Expression of gp160env

2.2.1. Mammalian Expression Vector pSV7d

pSV7c and pSV7d represent successive polylinker replacements. Thepolylinker in pSV7c was digested with BgIII and XbaI, and then ligatedwith the following linker to yield pSV7d:

  BglII EcoRI    SmaI  XbaI   BamHI Sa1I5′-GATCTCGAATTCCCCGGGTCTAGAGGATCCGTCGAC       AGCTTAAGGGGCCCAGATCTCCTAGGCACGTGGATC

2.2.2. Expression of tPA/gp160

In order to achieve optimal secretion of gp160 from mammalian tissueculture cells, the 5′ end of the coding sequence was modified to accepta heterologous signal sequence known to direct efficient secretion ofboth the homologous gene (human tissue plasminogen activator) anddeletion variants of this gene. van Zonnefeld et al. (1986) Proc. Natl.Acad. Sci. USA 83:4670.

A portion of the HIV env gene was excised with SacI and StuI (positions5555 and 6395) and was subcloned into the vector M13mp19 [Yanisch-Perronet al. (1985) Gene 33:103-109] between SacI and SmaI.Oligonucleotide-directed mutagenesis [Zoller et al. (1983) Methods inEnzymology 100:468-500] was used to create an NheI site at the junctionof the natural signal peptide and the mature envelope polypeptide usingthe following oligonucleotide:

5′-GATGATCTGTTCAGCTAGCGAAAAATTGTGG-3′

This mutagenesis changes cytosine-5867 to guanine and adenine-5868 tocytosine, thereby creating an NheI site and altering the codon forthreonine-30 to code for serine.

In parallel, the tPA gene was likewise mutagenized in M13 to place anNheI site near the carboxyl end of the tPA signal peptide. The followingsequences show the 5′ UT sequences and signal for wild type tPA leaderand for the NheI variant.

Wild-type sequence of the tPA signal:

5′ untranslated sequences

AGAGCTGAGATCCTACAGGAGTCCAGGGCTGGAGAGAAAACCTCTGCGAGGAAAGGGAAGGAGCAAGCCGTGAATTTAAGGGACGCTGTGAAGCAATCATG                                               MetGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCAspAlaMetLysArgGlyLeuCysCysValLeuLeuLeuCysGlyAlaValTTCGTTTCGCCCAGCCAGGAAATCCATGCCCGATTCAGAAGAGGAGCCAGAPheValSerProSerGlnGluIleHisAlaArgPheArgArgGlyAlaArg mature tPATCTTACCAAGTG SerTyrGlnVal

The NheI variant of the tPA signal:

5′ untranslated sequences

AGAGCTGAGATCCTACAGGAGTCCAGGGCTGGAGAGAAAACCTCTGCGAGGAAAGGGAAGGAGCAAGCCGTGAATTTAAGGGACGCTGTGAAGCAATCATG                                               MetGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCAspAlaMetLysArgGlyLeuCysCysValLeuLeuLeuCysGlyAlaValTTCGTTTCGCCCAGCGCTAGC PheValSerProSerAlaSer

Following mutagenesis and sequence verification, a 174 bp fragmentcontaining 99 bp of 5′ untranslated sequence and the signal sequencefrom tPA was excised from the tPA-containing M13 clone using SalI andNheI and fused to the 559 bp fragment containing the 5′ end of the envgene which was excised from the env-containing M13 clone with NheI andHindIII (contributed by the M13 polylinker) and these fragments weresubcloned into M13 mp18 between SalI and HindIII. This plasmid is calledM13tpaS.NheIenv.

The DNA and amino acid sequences of the tPA signal fused to the 5′ endof gp160 is:

 ...tPA signal...           5869 (amino acid 31 of env) Phe Val Ser ProSer Ala Ser Glu Lys Leu Trp Val Thr Val TTC GTT TCG CCC AGC GCT AGC GAAAAA TTG TGG GTC ACA GTT                     NheI

From this M13 subclone, M13tpaS.NheIenv, the DNA fragment containing theheterologous signal and 5′ untranslated sequences fused to the 5′ end ofthe env gene was excised using the XbaI site from the M13 polylinker andthe unique NdeI site found at position 5954 in the env gene. This 268 bpfragment was ligated with a 2506 bp NdeI-XhoI fragment, encoding theremainder of the env gene, from NdeI at position 5954 through the stopcodon at position 8344, and 116 additional bases to the XhoI site atposition 8460 and at the same time with vector pSV7d cleaved with XbaIand SalI in order to generate a fully functional gp160 gene in theproper orientation for expression from the SV40 early promoter. Thisexpression plasmid, called pSV7dARV160tpa, was used to transfect COS 7cells as described by Sompayrac et al. (1981) Proc. Natl. Acad. Sci. USA78:7575. Transfected cells were assayed for gp160 expression byimmunofluorescence as described in Section 2.1.3 above. Approximately 5%of the cells showed bright fluorescence when exposed to the HIV infectedhuman sera, and no cells stained with normal human serum. Positive seraemployed were obtained from the Interstate Blood Bank.

Permanent cell lines were obtained by transfection of CHO cells lackingdihydrofolate reductase (dhfr). CHO dhfr⁻ cells [Urlaub et al. (1980)Proc. Natl. Acad. Sci. USA 77:4216] were plated at a density of 5×10⁵ to10⁶ cells per 10 cm dish the day prior to transfection in nutrientmedium (F12 supplemented with 1.18 mg/ml Na₂CO₃, 292 ug/ml glutamine,110 ug/ml sodium pyruvate, 100 U/ml penicillin, 100 U/ml streptomycin,150 ug/ml proline and 10% fetal calf serum). Cells were transfectedusing the calcium phosphate coprecipitation method described by Grahamet al. (1973) Virology 52:456-467 with the gp160 expression plasmid,pSV7dARV160tpa; a plasmid bearing as a selectable marker, a dhfr genedriven by the adenovirus major late promoter, pAD-dhfr; and a plasmidbearing the human tissue plasminogen activator gene driven by the SV40early promoter, pSV7tpa3, to serve as an easily screened gene marker.Plasmid pSV7tpa3 is a mammalian cell expression vector containing thefull length tpa cDNA (with 99 bp 5′ untranslated sequences) cloned intothe SalI site of pSV7d, with the orientation such that the properreading frame is transcribed from the SV40 early promoter.

Samples were added to the dishes of cells fed with fresh medium andallowed to settle for 6 h in an incubator (5% CO₂, 95% air) at 37° C.Six hours later, the supernatants were aspirated, the cells rinsedgently with calcium and magnesium-free phosphate buffered saline(PBS-CMF), and the dishes exposed to 15% glycerol in HEPES-bufferedsaline as an adjuvant for 3.5 to 4 min. After rinsing, the medium on thecells was replaced with fresh F12 as described. Forty-eight hours afterthe addition of DNA to the cells, the cells were trypsinized and split1:20 into selective medium, Dulbecco's Modified Eagle Medium (DMEM)supplemented with 4.5 mg/ml glucose, 3.7 mg/ml Na₂CO₃, 292 ug/mlglutamine, 110 ug/ml sodium pyruvate, 100 U/ml penicillin, 100 U/mlstreptomycin, 150 ug/ml proline, and 10% dialyzed fetal calf serum.After growth in selective medium for 1-2 weeks, colonies appeared. Thesecolonies were assayed for production of tPA by thecasein-plasminogen-agar overlay assay. Granelli-Piperno et al. (1978) J.Exp. Med. 148:223-234. Individual clones were transferred to microwellplates and expanded to T75 flasks. Twenty-four cell lines that were thehighest tPA producers were screened by immunofluorescence for gp160expression as described for COS cells above and in Section 2.1.3. Ofthese, seven lines fluoresced brightly when reacted with HIV-positiveserum. These seven gp160-positive lines were pooled by mixing equalnumbers of each cell line and plating in 10 cm dishes at a cell densityof 1×10⁵ cells per dish. Replicate dishes were fed with DMEMsupplemented as described above with the addition of varying amounts ofmethotrexate (amethopterin, Sigma): 0.01, 0.02, 0.05, 0.1 and 0.2micromolar. After growth in the amplification medium containingmethotrexate for 2 to 3 weeks, colonies appeared on the 0.1 and 0.2micromolar methotrexate dishes. These colonies were transferred tomicrowell plates and expanded to T75 flasks.

Clones were screened and scored for increased tPA production. Lysates ofthe cells were prepared in order to examine any gp160 expressed andfound in the cell membranes, as is found in HIV infected humanlymphocytic lines. Lysates were prepared from cell monolayers by rinsingin cold PBS-CMF and adding lysis buffer consisting of 100 mM NaCl, 20 mMTris pH 7.5, 1 mM EDTA, 0.5% NP40, 0.5% sodium deoxycholate, 1% BSA, 1ug/ml pepstatin, 1 mM PMSF, and 17 ug/ml aprotinin. Lysates werecollected, incubated on ice for 10 min with occasional vortexing, spun 5min at 14000×g in a microcentrifuge, and supernatants transferred to newtubes for storage at −70° C. These samples were assayed by an ELISAspecific for envH derived from yeast (see Sections 4.1.2 and 4.1.5).

Immunoblot analysis was done by standard Western transfer of proteinsfrom 8% denaturing gels [Laemmli (1970) Nature 227:680-685] and analysiswith human immune sera or mouse monoclonal antibodies raised againstyeast-expressed envH and env-2 (see Sections 4.1.2 and 4.1.5). Theblocking solution is 0.3% Tween-20, 2% normal goat serum in phosphatebuffered saline, and all incubations and washes were done in thissolution. The human imune sera (Interstate Blood Bank) were diluted1:100, and Tago goat anti-human horse radish peroxidase (HRP) conjugatewas diluted 1:250. Development was with 4-chloro-1-napthol (BioRad).Mouse monoclonal antibody 98B3 [Stephans et al., in Viruses and HumanCancer, pp. 29-41 (R. C. Gallo et al., eds., 1987)] was used undilutedas ascites fluid or diluted to 50 ug/ml from a stock of ammonium sulfatepurified 1 g. Goat anti-mouse HRP conjugate (Tago) was diluted 1:200.

ELISA analysis was done using the cellular lysates. Plates were coatedwith mouse monoclonal IgM antibody 95C9 [Stephans et al., supra] at aconcentration of 250 ng per 100 microliters per well in PBS. After 2 hat 37° C. or overnight at 4° C., the plates were washed 6 times in washbuffer (0.137 M NaCl, 0.05% Triton X-100) and the samples added to thewells in casein diluent (100 mM sodium phosphate, 0.1% casein, 1 mMEDTA, 1% Triton X-100, 0.5 M NaCl, 0.01% thimerosol pH7.5), and plateswere incubated overnight at 4° C. and 4 h at 37. Samples were aspirated,then plates were washed 6 times as above. Rabbit anti-env-2 polyclonalserum [Barr et al. (1987) Vaccine 5:90-101] is diluted 1:100 in thediluent above and added to the plates (100 microliters per well). Plateswere incubated for 1 h at 37° C., and then aspirated and washed asdescribed above. Goat anti-rabbit HRP conjugate (Tago) was diluted1:1000 in the diluent buffer, and 100 microliters were added to eachwell. Plates were incubated 1 h at 37. Samples were aspirated and theplates were washed as described above. Plates were developed with ABTScolor reagent and H₂O₂ for 30 min at 37. ABTS was dissolved in citratebuffer and 100 microliters per well were added to the plate. Thereaction was stopped by adding 50 ul per well 10% SDS, and the plateswere read at 415 nm, 600 nm reference beam. Env-2 was used as a standardusing serial 2-fold dilutions starting with 100 ng/ml to 11 places.

Six CHO clones positive by the ELISA assay were amplified in a pool inmethotrexate as described above, except that after isolation of coloniesfrom the first round of amplification, cells were again pooled and setup for a consecutive round of amplification. For the first round,methotrexate levels were 0.25, 0.5, 0.75, 1.0, 1.25, and 1.75micromolar. Colonies from the 0.5 and 0.75 micromolar dishes were pooledand set up in 0.75, 1.0, 2.0, 3.0, 4.0, and 5 micromolar. Clones wereisolated from 3, 4, and 5 micromolar levels, expanded and assayed forgp160 production by the ELISA. Increases in expression were observed forthese clones as compared to the clones grown in 0.1 and 0.2 micromolar

In addition to the two types of recombinant phage containing HIV DNAdescribed above, phage was obtained that (1) possessed the left half ofthe viral genome from the EcoRI site in viral DNA extending intoflanking cell DNA [λARV-2(8A)] and (2) phage that had the right half ofthe viral genome [λARV-2(7D)] from the EcoRI site in viral DNA extendinginto flanking cell DNA. The four types of recombinant phage DNAstructures are predicted from the analysis of viral DNA observed ininfected cells. Maps of restriction enzyme sites are shown in FIGS. 1-3.The data for these maps were compiled from studies of proviral DNA ininfected cells, from characterization of recombinant phage DNA, and frompreliminary DNA sequence information. Bacteriophages λARV-2(7D) (right)and λARV-2(8A) (left) were deposited at the ATCC on Oct. 26, 1984 andgiven Accession Nos. 40143 and 40144, respectively.

To validate the cloned HIV DNA, a radioactive probe was prepared fromtwo regions of the cloned HIV genome that represent about 70% of thegenome and this probe was used to detect HIV DNA in restriction enzymedigests of DNA from infected cells. Whole-cell DNA was prepared fromHUT-78 cell cultures infected with HIV-1 strains ARV-2, ARV-3 and ARV-4,and analyzed by digestion with restriction enzymes as described inSection 1.3. A probe to cloned ARV-2 DNA was prepared as follows: DNAfrom recombinant phage λ-7A (FIG. 3) was digested with SacI or with amixture of SacI and KpnI and digestion products were electrophoresed in1% agarose gels (low-melting agarose); the 3.8 kb DNA fragment from thedigestion with SacI and the 3.1 kb DNA fragment from the digestion withSacI and KpnI (FIG. 3) were eluted, pooled, denatured by boiling inwater for 2 methotrexate, and this expression level represents animprovement over the levels of gp160 in HIV-infected HUT cells.

2.3 Expression of gp120env

2.3.1. Expression of Engineered gp120 in pS7Vd

In order to express a secreted form of the envelope polypeptide inmammalian cells, the env gene was modified by in vitro mutagenesis toeliminate any potential transmembrane domains and to provide a stopcodon following the processing site between the gp120 and gp41 domainsof the gp160 protein. This mutagenesis was accomplished by subcloningthe fragment which encodes the env gene from clone pSV7c/env (Section2.1.2.) by excising with HindIII and XhoI (positions 5582 to 8460) andinserting the 2.8 kb fragment into M13 mp19 previously digested withHindIII and SalI. A 37 bp oligonucleotide of the following sequence wasused to alter the sequence at the gp120/gp41 processing site at position7306 to encode 2 stop codons and two restriction endonuclease sites.

5′-GAACATAGCTGTCGACAAGCTTCATCATCTTTTTTCT-3′

The sequence of the wild-type gene and the mutant are shown below:

Wild-type sequence:                                    Processing sitePosition:   7288    7294    7300                    7318 Amino acid: ValGln Arg Glu Lys Arg Ala Val Gly Ile Val acid: DNA:        GTG CAG AGAGAA AAA AGA GCA GTG GGA ATA GTA acid: Val Gln Arg Glu Lys Arg Ala ValGly Ile Val DNA:        GTG CAG AGA GAA AAA AGA GCA GTG GGA ATA GTAgp120 mutant: Position:   7288    7294    7300    7306          7317Amino acid: Val Gln Arg Glu Lys Arg OP  OP DNA:        GTG CAG AGA GAAAAA AGA TGA TGA AGC TTG TCG AC Restrictionsite:                         HindIII SalI

Following mutagenesis, the gene was engineered for optimal secretioninto the medium utilizing the same procedure that was used for gp160.The 268 bp XbaI-NdeI fragment containing the heterologous 5′untranslated sequences and signal sequences from human tPA fused to the5′ end of env was excised from the M13 clone M13tpaS.NheIenv describedin Section 2.2.2. This fragment was ligated with a 1363 bp NdeI-SalIfragment encoding the remainder of the gp120 coding region which wasisolated from the gp120 mutant (positions 5954 and 7317) describedabove, and both fragments were inserted into the vector pSV7d previouslydigested with XbaI and SalI.

This plasmid, pSV7dARV120tpa, has a gene encoding gp120 that endsprecisely at the processing site between gp120 and gp41, Arginine-509,expressed from the SV40 early promoter. Plasmid pSV7dARV120 was used totransfect COS7 cells as described in Section 2.2.2. Transfected cellswere assayed for expression and secretion of gp120 into the culturemedium by immunoblot analysis of conditioned cell medium from thetransfected cells. Immunoblot analysis was done using both HIV-positivehuman serum and mouse monoclonal antibodies described in Section 2.2.2.Using both antibody sources, the gp120 transfected cells secreted intothe medium a polypeptide of the size of gp120 that reacted with theseimmunological reagents, while mock transfected or pSV7dARV160tpatransfected COS cell supernatants showed no such polypeptide. Theseresults are consistent with the production of authentic gp120 in COScells.

2.3.2. Expression of gp120env Using CMV IE-1 Promoter

In an effort to improve the expression of gp120 in COS and othermammalian cell types, the gp120 coding region was excised frompSV7dARV120tpa using SalI and inserted into the unique SalI site of theCMV IE-1 expression vector pCMV6a (described below) and screened forinsertion in the proper orientation for expression from the CMV IE-1promoter. The resulting plasmid, pCMV6ARV120tpa, was used to transfectCOS 7 cells as described in Sections 2.2.2. and 2.3.1. The conditionedcell medium from the transfected cells was assayed by immunoblot and byELISA, as described in Sections 2.2.2. and 2.3.1. TheCMV6ARV120tpa-transfected cells expressed gp120 at levels of 60-250ng/ml and the pSV7ARV120tpa-transfected cells expressed less than 0.5ng/ml. These results indicate a significant increase in expression ofthe gp120 polypeptide using the CMV expression plasmid relative to theSV40 expression plasmid described in Section 2.3.1, 50 to 100-fold inmagnitude as measured by the ELISA.

Plasmid pCMV6a is a mammalian cell expression vector which contains thetranscriptional regulatory region from human cytomegalovirus immediateearly region, HCMV IE1. The plasmid contains the SV40 polyadenylationregion derived from pSV7d (Section 2.2.1.) as a 700 bp PvuI-SalIfragment; the SV40 origin of replication derived for pSVT2 [Meyers etal. (1981) Cell 25:373-384; Rio et al. (1983) Cell 32:1227-1240] as a1.4 kb PvuI-EcoRI (filled in with Klenow fragment); and the HCMV IE1promoter as a 1.7 kbp SspI-SalI fragment derived from a subclone of thehuman cytomegalovirus Towne strain). The HCMV IE1 promoter regioncontains the region encoding the first exon (5′ untranslated), the firstintron and the start of the second exon (where the SalI site was createdby in vitro mutagenesis). The map of pCMV6a is shown in FIG. 29.

2.4. Expression of Gag Peptides

The ARV-2 DNA coding for the gag region was excised from a recombinantphage (7A; FIG. 3) by digestion with KpnI and SacI and gel purificationof a fragment of about 3200 bp. This gag containing fragment was clonedinto KpnI and SacI digested pSV-7c/envΔES (described below) to yieldpSV-7c/gag. The recombinant vector was used to transfect Cos cells.Expression of gag protein was detected as previously described for theenv protein (Section 2.1). About 5 percent of the cells transfected withSV40 vector containing the ARV-2 gag fragment showed brightimmunofluorescence when incubated with the AIDS sera and a secondfluoresceinated goat anti-human antisera. Cells transfected with acontrol plasmid containing only SV40 vector sequences did not show anyimmunofluorescence with any antisera.

Plasmid pSV-7c/envΔES was constructed as follows: Plasmid pSV-7c/env(described in section 2.1.2) was digested with EcoRI and SacI. A linkerof the following sequence, which regenerates both the EcoRI and SacIsites and provides for a BamHI site between them was ligated to thelinearized vector.

  AATTCGGATCCGAGCT       GCCGAGGC EcoRI  BamHI  SacIThis vector was digested with KpnI and SacI, the fragment correspondingto linearized plasmid was purified by gel electrophoresis and ligated tothe 3200 bp KpnI-SacI fragment containing the gag region.2.5 Expression of gag-env Fusion Protein

A mammalian cell expression vector containing a fused sequence of nt 225to nt 1650 encoding for a gag region and nt 5957 to nt 8582 for an envregion (FIG. 5) was constructed as follows. Plasmid pSV-7c/gag(described in Section 2.4) was digested with BglII. The overhangs werefilled in with reverse transcriptase, and the resulting two fragmentswere subsequently digested with SacI. The 1390 SacI-BglII bp fragmentcoding for gag was purified by gel electrophoresis and cloned intopSV-7c/env (Section 2.1.2) prepared as follows.

Vector pSV-7c/env was digested with NdeI which cuts 59 codons downstreamfrom the N-terminal ATG of env. Ends were filled in with reversetranscriptase and the linearized vector was digested with SacI. TheSacI-NdeI vector fragment was purified by gel electrophoresis andligated to the gag fragment obtained as indicated above. The ligationmixture was used to transform HB101, and plasmid pSV-7c/gagΔenv was thusobtained.

Plasmid pSV-7c/gagΔenv was used to transform Cos cells using theprocedure described in Section 2.1.2 above. Expression was detected byimmunofluorescence using an AIDS patient serum and following theprocedure described previously (Section 2.1.3). About 5 cells in 100showed bright immunofluorescence with the immune serum. No cells stainedwith control normal human serum.

3. Expression of HIV Polypeptides in Bacteria

3.1. Expression of p25gag

3.1.1. Host-Vector System

The p25gag protein is synthesized by E. coli strain D1210 transformedwith plasmid pGAG25-10. Plasmid pGAG25-10 is a pBR322 derivative whichcontains the sequence coding for p25gag under transcriptional control ofa hybrid tac promoter [De Boer et al. (1983) Proc. Natl. Acad. Sci. USA80:21-25] derived from sequences of the trp and the lac UV5 promoters.Expression of p25gag is induced in bacterial transformants withisopropylthiogalactoside (IPTG).

E. coli D1210, a lac-repressor overproducing strain, carries thelacI^(q) and lacY⁺ alleles on the chromosome but otherwise is identicalto E. coli HB101 (F⁻ lacI⁺, lacO⁺, lacZ⁺, lacY⁻, gal⁻, pro⁻, leu⁻, thi⁻,end⁻, hsm⁻, hsr⁻, recA⁻, rpsL⁻) from which it was derived.

3.1.2. Construction of pGAG25-10.

Plasmid pGAG25-10 was constructed by cloning a 699 bp DNA fragmentcoding for p25gag into plasmid ptac5, according to the scheme shown inFIG. 7. The vector ptac5 is a pBR322 derivative which contains the tacpromoter, Shine Delgarno sequences, and a polylinker as a substitutionof the original pBR322 sequences comprised between the EcoRI and PvuIIrestriction sites.

The 699 bp DNA fragment codes for the complete p25gag protein (aminoacid residues 136 to 365 as numbered in FIG. 5), the only differencebeing that a methionine was added as the first amino acid in pGAG25-10to allow for translational initiation. This change, as well as otherchanges in nucleotide sequence as indicated below, were achieved byusing chemical synthesis of parts of the DNA fragment. The DNA fragmentalso includes two stop codons at the 3′ end of the sequence.

FIG. 8 shows the nucleotide sequence cloned in pGAG25-10 and the aminoacid sequence derived from it. DNA sequences that are not underlined inthe figure were derived directly from the λARV-2(9B) DNA. All othersequences were chemically synthesized or derived from vector ptac5.Changes were introduced in this DNA sequence, with respect to theoriginal DNA, to create or delete restriction sites, to add a methionineprior to the isoleucine (second residue of p25) or to include stopcodons after the last codon of p25gag. However, as previously indicated,all changes in the DNA sequence, except those in the first codon, do notalter the amino acid sequence of p25gag.

3.1.3. Transformation and Expression of gag Peptide

E. coli D1210 cells are made competent for transformation following astandard protocol [Cohen et al. (1982) Proc. Natl. Acad. Sci. USA69:2110]. Transformation is performed as indicated in the protocol with25-50 ng of pGAG25-10. The transformation mix is plated on agar platesmade in L-Broth containing 100 μg/ml ampicillin. Plates are incubatedfor 12 h at 37° C.

Single ampicillin resistant colonies are transferred into 1 ml L-Brothcontaining 100 μg/ml ampicillin and grown at 37° C. Expression of p25gagprotein is induced by adding 10 μl of 100 mM IPTG (Sigma) to a finalconcentration of 1 mM followed by incubation at 37° C. for 2 h.

Cells from 1 ml of induced cultures are pelleted and resuspended in 100μl Laemmli sample buffer. After 3 cycles of boiling and freezing,portions of resultant lysates are analyzed on standard denaturingacrylamide gels. Proteins are visualized by staining with Coomassieblue.

The extent of expression is initially determined by appearance of newprotein bands for induced candidate samples compared with control.Proteins of molecular weights expected for the genes expressed comprised2%-5% of total cell protein in the highest expressing recombinants asdetermined by visual inspection with reference to a standard protein ofknown amount.

Authenticity of the expressed proteins is determined by standard Westerntransfer of proteins to nitrocellulose and analysis with appropriatehuman or rabbit immune sera or mouse monoclonal antibodies or bycomparing ELISA assays of purified recombinant and natural proteinsusing human immune sera from AIDS patients (see Section 3.1.6).

3.1.4. Fermentation Process

3.1.4.1. Preparation of Master Seed Stock

Transformant cells from a culture expressing high levels (3%) of p25gagare streaked onto an L-Broth plate containing 100 μg/ml ampicillin andthe plate is incubated overnight at 37° C. A single colony is inoculatedinto 10 ml of L-Broth, 100 μg/ml ampicillin and grown overnight at 37°C. An aliquot is used to verify plasmid structure by restriction mappingwith SalI and PstI. A second aliquot is used to induce expression ofp25gag and the rest of the culture is made 15% glycerol by adding ¼volume of 75% sterile glycerol. Glycerol cell stocks are aliquoted in 1ml and quickly frozen in liquid nitrogen or dry-ice ethanol bath. Thesemaster seed stocks are stored at −70° C.

3.1.4.2. Fermenter Inoculum

The master seed stock is scraped with a sterile applicator which is usedto streak an L-Broth plate containing 100 μg/ml ampicillin. Singlecolonies from this plate are used to inoculate 20-50 ml of L-Broth/amp,which is incubated at 37° C. overnight.

An aliquot of the overnight culture is used to inoculate larger volumes(1-6 liters) of L-Broth/amp. Cells are incubated at 37° C. overnight andreach an O.D.₆₅₀ of approximately 5 prior to use as inoculum for thefermenter run.

3.1.4.3. Fermentation and Harvest

Fermenters (capacity: 16 liters) containing 10 l of L-Broth and 1 ml ofantifoam are inoculated with 100-500 ml from the inoculum culture. Cellsare grown at 37° C. to an O.D. of about 1. Expression of p25gag isinduced by addition of 100 ml of an IPTG solution (100 mM) to yield a 1mM final concentration in the fermenter. Cells are grown for 3additional hours and subsequently harvested using continuous flowcentrifugation. At this step cells may be frozen and kept at −20° C.until purification of p25gag proceeds. Alternatively, 250 l fermentersare inoculated with 1-5 l from the inoculum culture. Growth, induction,and harvest are as indicated before.

3.1.5. Purification of p25gag

Frozen E. coli cells are thawed and suspended in 2.5 volumes of lysisbuffer (0.1M sodium phosphate (NaPi), pH 7.5, 1 mM EDTA, 0.1 M NaCl).Cells are broken in a non-continuous system using a 300 ml glass unit ofa Dyno-mill at 3000 rpm and 140 ml of acid-washed glass beads for 15min. The jacketed chamber is kept cool by a −20° C. ethylene glycolsolution. Broken cells are centrifuged at 27,000×g for 25 minutes toremove debris and glass beads. The supernatant is recovered and kept at4° C.

The cell extract is made 30% (NH₄)₂SO₄ by slowly adding the ammoniumsulfate at 4° C. The extract is stirred for 10 min after the finalconcentration is achieved, followed by contrifugation at 27,000×g for 20min. The pellet is resuspended in 1 M NaCl, 1 mM EDTA, 1% Triton X-100,and 5% SDS, and then boiled for 5 min.

The fraction obtained by selective precipitation is submitted to gelfiltration using a G50 Sephadex column equilibrated in 0.03 M NaPi, pH6.8. Chromatography is developed in the same solution. Fractions arecollected and absorbance at 280 nm is determined. Protein-containingfractions are pooled and characterized by protein gel electrophoresis,Western analysis, and ELISA.

3.1.6. Characterization of Recombinant p25gag

3.1.6.1. Protein Gel Electrophoresis

SDS-polyacrylamide gel analysis (10%-20% gradient gels) of proteins frompGAG25-containing cells and control cells indicated that varying levelsof a protein of a molecular weight of about 25,000 were specificallyinduced in cells containing p25gag expression plasmids afterderepression of the tacI promoter with IPTG. Identity of the p25gag geneproduct was confirmed by both an enzyme-linked immunosorbent assay(ELISA; see Section 3.1.6.3) and Western immunoblot analysis (seeSection 3.1.6.2) using both AIDS patient serum and a monoclonal antibodyto viral p25gag.

3.1.6.2. Western Analysis

Samples were electrophoresed under denaturing conditions on a 10%-20%polyacrylamide gradient gel. Samples were electroblotted ontonitrocellulose. The nitrocellulose paper was washed with a 1:250dilution of AIDS patient reference serum (EW5111, obtained from P.Feorino; Centers for Disease Control, Atlanta, Ga.) and then with a1:500 dilution of HRP-conjugated goat antiserum to human immunoglobulin(Cappel, No. 3201-0081). Alternatively, the nitrocellulose was washedwith undiluted culture supernatant from 76C, a murine monoclonalantibody to HIV-1 p25gag, and then with a 1:500 dilution ofHRP-conjugated goat antiserum to mouse immunoglobulin (TAGO, No. 6450).The substrate for immunoblots was HRP color development reagentcontaining 4-chloro-1-naphthol.

The p25gag protein reacted with both AIDS patient reference serum andwith the monoclonal antibody, while it shows no reactivity with thenon-immune serum.

3.1.6.3. ELISA Comparison of Recombinant and Native p25gag

The reactivity of sera with the purified p25gag (recombinant and native)was assayed by coating wells of microtiter plates with 0.25 μg/ml ofpurified antigen, adding dilutions of test sera, followed by a 1:1000dilution of HRP-conjugated goat antiserum to human immunoglobulin.Finally, the wells received substrate solution (150 μg/ml2,2′-azino-di-[3-ethylbenzyl-thiazoline sulfonic acid], 0.001% H₂O₂, 0.1M citrate pH 4). The reaction was stopped after incubation for 30 min at37° C. by the addition of 50 μl/well of 10% SDS. The absorbance was readon a Flow Titertech ELISA reader at 414 nm. Samples were assayed induplicate beginning at a dilution of 1:100 and by serial 2-folddilutions thereafter.

As an example, a single sample of HIV antibody-positive serum and normalhuman serum were each titrated on both recombinant and native p25gag.The antibody-positive serum reacted equally with viral and recombinantantigen. There was no reaction seen with serum from a normal individual.

The reactivity of purified recombinant p25gag to various sera was thencompared to that of natural p25gag protein purified by preparativepolyacrylamide gel electrophoresis in an ELISA assay. For comparisons,assays were also done using disrupted gradient purified virus (5 μg/ml)as antigen. The table below summarizes the results of assays on 8 AIDSsera that scored positive in the assay with disrupted virus and 6 normalsera that were negative in the disrupted virus assay.

SERUM ELISA ASSAY TITER^(a) NUMBER Disrupted Virus Recomb. p25gag Viralp25gag Group I: Sera Scoring As Positive in Virus ELISA^(b) 1 51,2003,125 3,125 5 12,800 25 25 6 12,800 625 625 7 12,800 3,125 3,125 825,600 15,625 15,625 9 12,800 625 625 13 800 125 125 18 3,200 625 625GroupII: Sera Scoring Negative in Virus ELISA^(b) 15  —^(c) — — 16 — — —19 — — — 21 — — — 26 — — — 33 — — — ^(a)Reported as the reciprocal ofthe serum dilution that gave a signal equivalent to 50% of the maximum.^(b)Results were confirmed by immunofluorescence and immunoblotting asdescribed previously. ^(c)No detectable signal at a 1:25 serum dilution.

These results show that p25gag purified from bacteria behavesidentically to similarly purified p25gag from AIDS virus in an ELISA ofthe eight AIDS patient sera. The results of the ELISA show that there isa wide variation in the levels of anti-p25gag antibodies and suggeststhat antibodies to some virus-encoded proteins may not be detected usingconventional virus-based assay systems.

3.2. Expression of p16gag

The sequence shown in FIG. 10 and coding for the p16gag protein waschemically synthesized [Warner et al. (1984) DNA 3:401] usingyeast-preferred codons. The blunt-end to SalI fragment (381 bp) wascloned into PvuII-SalI digested and gel-isolated ptac5 (see Section3.1). The resulting plasmid, pGAG16, was used to transform E. coli D1210cells, as in Section 3.1.3. Expression was induced with IPTG, andproteins were analyzed by polyacrylamide gel electrophoresis and Westernanalysis. A band of about 16,000 daltons was induced by IPTG in thetransformed cells. This protein showed reactivity in Western blots withimmune sera from AIDS patients. No reactivity was observed with serafrom normal individuals.

3.3 Expression of Fusion Protein p41gag

A fusion protein of the p25gag and p16gag proteins of ARV-2, designatedp41gag, was synthesized in E. coli strain D1210 transformed with plasmidpGAG41-10. pGAG41-10 was constructed from plasmid pGAG25-10 (see Section3.1.2) as shown in FIG. 7 by inserting an SphI-HpaI fragment from theARV-2 genome containing the sequences from the C-terminal p16gag portionof the p53gag precursor polyprotein and part of the p25gag proteinbetween the SphI and BamHI sites of pGAG25-10. The coding strand of theDNA sequence cloned in pGAG41-10 is shown in FIG. 9. Transformation andinduction of expression were effected by the procedures described above.The cells were treated and the p41gag protein was visualized onCoomassie-stained gel as described above. The approximate molecularweight of the observed protein was 41,000 daltons. The protein reactedwith AIDS sera and monoclonal antibody to p25gag in Western and ELISAanalyses carried out as above.

3.4. Expression of p31pol

3.4.1. Host-Vector System

The C-terminal region of the polymerase gene (p31pol) is synthesized byE. coli strain D1210 transformed with plasmid pTP31.2. Plasmid pTP31.2is a pBR322 derivation which contains the sequence coding for p31 undertranscriptional control of the hybrid tac promoter (described in Section3.1). Expression of p31 is induced in bacterial transformants by IPTG.

3.4.2. Construction of pTP31.2

3.4.2.1. Construction of M13 Template 01100484

A 4.87 kb DNA fragment was isolated from a KpnI digest of ARV-2 (9b)containing the 3′ end of the pol gene, orf-1, env and the 5′ end oforf-2, that had been run on a 1% low melting point agarose (Sea-Pack)gel and extracted with phenol at 65° C., precipitated with 100% ethanoland resuspended in TE. Eight μl of this material were further digestedwith SstI for 1 h at 37° C. in a final volume of 10 μl. After heatinactivation of the enzyme, 1.25 μl of this digest were ligated to 20 ngof M13mp19 previously cut with KpnI and SstI, in the presence of ATP andin a final volume of 20 μl. The reaction was allowed to proceed for 2 hat room temperature. Five μl of this mixture were used to transformcompetent E. coli JM101. Clear plaques were grown and single-strandedDNA was prepared as described in Messing & Vieira, (1982) Gene19:269-276. One of these plaques containing the 1848 bp fragment wasdesignated 01100484.

3.4.2.2. In Vitro Mutagenesis of 01100484

The DNA sequence in 01100484 was altered by site-specific mutagenesis togenerate a restriction site recognized by NcoI (CCATGG). Anoligodeoxynucleotide that substitutes the A for a C at position 3845(FIG. 5) and changes a T for an A at position 3851 (FIG. 5) wassynthesized using solid phase phosphoramidite chemistry. Both of thesechanges are silent in terms of the amino acid sequence, and the secondone was introduced to decrease the stability of the heterologousmolecules. The oligomer was named ARV-216 and has the sequence:5′-TTAAAATCACTTGCCATGGCTCTCCAATTACTG and corresponds to the noncodingstrand since the M13 derivative template 01100484 is single-stranded andcontains the coding strand. The 5′ phosphorylated oligomer was annealedto the 01100484 M13 template at 55° C. in the presence of M13 sequencingprimer, 50 mM Tris-HCl pH 8, 20 mM KCl, 7 mM MgCl₂ and 0.1 mM EDTA. Thepolymerization reaction was done in 100 μl containing 50 ng/μl DNAduplex, 150 μM dNTPs, 1 mM ATP, 33 mM Tris-acetate pH 7.8, 66 mMpotassium acetate, 10 mM magnesium acetate, 5 mM DTT, 12.5 units of T4polymerase, 100 μg/ml T4 gene 32 protein and 5 units of T4 DNA ligase.The reaction was incubated at 30° C. for 30 min and was stopped by theaddition of EDTA and SDS (10 mM and 0.2% respectively, finalconcentration).

Competent JM101 E. coli cells were transformed with 1, 2, and 4 μl of a1:10 dilution of the polymerization product and plated into YT plates.Plaques were lifted by adsorption to nitrocellulose filters anddenatured in 0.2 N NaOH, 1.5 M NaCl, followed by neutralization in 0.5 MTris-HCl pH 7.3, 3 M NaCl and equilibrated in 6×SSC. The filters wereblotted dry, baked at 80° C. for 2 h and preannealed at 37° C. in 0.2%SDS, 10× Denhardt's, 6×SSC. After 1 h, 7.5 million CPM of labeledARV-216 were added to the filters and incubated for 2 additional h at37° C. The filters were washed in 6×SSC at 42° C. for 20 min, blot-driedand used to expose film at −70° C. for 1 h using an intensifying screen.Strong hybridizing plaques were grown and single-stranded DNA wasprepared from them and used as templates for sequencing. Sequencingshowed that template 01021785 contains the NcoI site as well as thesecond substitution mentioned above.

A second oligomer was synthesized to insert sites for SalI and EcoRIimmediately after the termination codon of the pol gene (position 4647,FIG. 5). This oligomer was called ARV-248 and has the sequence:5′-GGTGTTTTACTAAAGAATTCCGTCGACTAATCCTCATCC. Using the template 01020785,site specific mutagenesis was carried out as described above except thatthe filter wash after the hybridization was done at 65° C. As above, 8strong hybridizing plaques were grown and single-stranded DNA wassequenced. The sequence of template 01031985 shows that it contains therestriction sites for NcoI, SalI, and EcoRI as intended.

3.4.2.3. Isolation of DNA Fragments Containing p31

Replicative form (RF) of the 01031985 template was prepared by growing 6clear plaques, each in 1.5 ml of 2×YT at 37° C. for 5 h. Double-strandedDNA was obtained as described by Maniatis et al., supra, pooled andresuspended in 100 μl final volume. Ten μl of RF were digested with NcoIand EcoRI in a final volume of 20 μl. This fragment was used for directp31 expression in bacteria. An additional 20 μl of RF were cut with NcoIand SalI in 40 μl. This fragment was used for p31 expression in yeast.The samples were run on a 1% low melting point agarose (Sea-Pack) geland the DNAs were visualized by fluorescence with ethidium bromide. The800 bp bands were cut and the DNAs were extracted from the gel asmentioned above and resuspended in 10 μl of TE. The fragments werecalled ARV248NR#2 and ARV248NL, respectively.

3.4.2.4. Cloning of p31 into Plot 7

The vector plot #7 (3 μg) [Hallewell et al. (1985) Nucl. Acid Res.13:2017-2034] was cut with NcoI and EcoRI in 40 μl final volume and theenzymes were heat-inactivated after 3 h. Two μl of this digest weremixed with 2 μl of ARV248NR#2 and ligated in 20 μl in the presence ofATP and T4 DNA ligase at 14° C. overnight, and 10 μl of this mixturewere used to transform competent D1210 cells. Colonies resistant to 2 mMIPTG and 100 μg/ml ampicillin were selected and supercoiled DNA wasextracted from each of them. The DNAs were then restricted with NcoI andEcoRI and analyzed by agarose gel electrophoresis. Clones with theappropriate 800 bp insert were selected for further use. They aredesignated pRSP248 numbers 3 and 4.

3.4.2.5. Construction of pTP31

The NcoI site introduced into 01100485 is 52 bp downstream from theputative start of p31. Three oligomers were synthesized as above thatcode for the first 17 amino acids of p31 and generate a cohesive NcoIend at the 3′ end of the molecule. The 5′ end of the molecule has beenextended beyond the initiation codon to include a ribosome binding site.The oligomers that were synthesized have the sequences:

ARV-221            CCCC     C  C 5′AGGXAACAGAAAAATGATAGATAAGGCACAAGAA           TTTT     T ARV-222 5′GAACATGAGAAATATCACAGTAATTGGAGAGC ARV-2233′CGTGTTCTTCTTGTACTCTTTATAGTGTCATTAACCTCTCGGTAC

One hundred fifty picomoles each of dephosphorylated ARV-221,phosphorylated ARV-222 and ARV-223 were ligated to 20 μg of pRSP248previously cut with NcoI, at 14° C. for 18 h in a final volume of 62 μl.After phenol extraction and ethanol precipitation, the DNA wasresuspended in 40 μl H₂O and incubated with 15 units of Klenow fragmentin the presence of 0.5 mM dNTPs for 1 h at room temperature. The samplewas phenol extracted, ethanol precipitated, resuspended in 40 μl H₂O,and digested with EcoRI. The DNA was then run on a low melting pointagarose gel and the fragment of about 820 bp was extracted as describedabove and resuspended in a final volume of 20 μl of H₂O. Afterphosphorylating the ends, 5 μl of the sample were incubated for 18 h at14° C. with 150 ng of plot7 that had been cut with PvuII and EcoRI andits ends dephosphorylated, in the presence of T4 DNA ligase, ATP and ina final volume of 31 μl. Five μl of ligation product were used totransform RR1ΔM15. Clones resistant to 100 μg/ml of ampicillin wereselected and supercoiled DNA was extracted from them. The DNAs weredigested with NcoI and EcoRI and resolved on a 1% agarose gel. Colonieswith the appropriate size insert were obtained and named pTP31. The p31sequence contained in the insert is shown in FIG. 12. Underlinedsequences were chemically synthesized. Others were derived from DNA.

3.4.3. Screening of Transformants

Bacterial transformants containing either the vector alone, or thevector with the p31 DNA (pTP31.2) were grown in L-Broth with 0.02%ampicillin to an OD₆₅₀ of 0.5. Cultures were induced by the addition ofIPTG to a final concentration of 2 mM and grown for 3 more hours.Bacteria from 1 ml cultures were pelleted and resuspended in 200 μl ofgel sample buffer. The cells were disrupted by three cycles of freezingand thawing, boiled, and the extracts loaded onto 12.5%polyacrylamide-SDS minigels. Proteins were electrophoresed andtransferred to nitrocellulose by electro-blotting. The nitrocellulosefilters were reacted with serum EW5111 (diluted 1:100; positivereference serum from the CDC that reacts strongly with viral p31), horseradish peroxidase-conjugated goat anti-human IgG and HRP substrate. Aprominent band at ˜30,000 d and several lower molecular weight specieswere seen in gels of extracts from transformants with the p31 DNA, butnot in extracts from bacteria transformed with the vector alone.

3.4.4. Characterization of Recombinant Protein

Lysozyme-NP40 extracts were prepared from bacteria transformed withpTP31.2 or vector alone. Five ml cultures were grown, the cells pelletedand resuspended in 1 ml of 50 mM Tris-HCl pH 8, 0.5 mM EDTA, 1 mg/mllysozyme and incubated at 0° C. for 15 min NaCl, MgCl₂, and NP40 wereadded to final concentrations of 0.4, 5 mM and 0.5% respectively, mixedand incubated with DNAse I (100 μg/ml) at 0° C. for 30 min. When EW5111serum (diluted 1:100) was preincubated with a 1:10 dilution of the cellextracts from bacteria transformed with pTP31.2, prior to reaction witha virus blot, the viral p31 band was completely eliminated, whilereactivity with other viral proteins remained unaffected. In contrast,extracts from bacteria transformed with the vector alone did not absorbout the p31 reactive antibodies. The viral p31 protein is thus theproduct of the C-terminal or endonuclease region of the pol gene ofHIV-1.

3.5. Expression of SOD-p31 Fusion Protein

In order to generate a fused protein SOD-p31 that can be expressed in E.coli, 2 μl of the fragment ARV248NL were ligated to 100 ng of aNcoI-SalI digest of pSODCF2 [Steimer et al. (1986) J. Virol. 58:9-16], aplasmid that contains the human SOD coding region under the regulationof the tacl promoter, in 20 μl final volume for 18 h at 14° C. CompetentD1210 cells were transformed with 5 μl of the ligation mix and colonieswere selected on L-broth ampicillin plates. Colonies with the correctinsert were called E. coli D1210 (pTSp31). The expression of the fusionprotein was analyzed in the same manner as the direct expression of p31.The analysis showed production of large amounts of a protein of about 47kd that is immunoreactive with EW5111 but not with normal human sera.Further characterization of the recombinant protein is described inSection 4.3.3.3.

3.6. Expression of SOD-env5b Fusion Protein

3.6.1. Host-Vector System A hybrid protein consisting of humansuperoxide dismutase (SOD) fused to a viral envelope (env-5b) protein issynthesized by E. coli strain D1210 transformed with plasmid pSOD/env5b.Plasmid pSOD/env5b is a bacterial expression vector which contains thesequence coding for human SOD [Hallewell et al. (1985) Nucl. Acid. Res.13:2017] fused to the env-5b region of the viral env gene[Sanchez-Pescador et al. (1985) Science 227:484] as well as pBR322sequences including the ampicillin resistant (amp^(R)) gene.

Expression of SOD/env-5b fusion protein is non-constitutive and it isunder transcriptional control of a hybrid tac promoter [De Boer et al.(1983) Proc. Natl. Acad. Sci. USA 80:21-25] derived from sequences ofthe trp and the lac UV5 promoters. Expression of SOD/env-5b is inducedin bacterial transformants with IPTG.

E. coli D1210, a lac-repressor overproducing strain, carries the lacIand lacY ⁺ alleles on the chromosome but otherwise is identical to E.coli HB101 (F⁻ lacI⁺, lacO⁺, lacY⁻, gal⁻, pro⁻, leu⁻, thi⁻, end⁻, hsm⁻,hsr⁻¹, recA⁻, rpsL⁻) from which it was derived [Sadler et al. (1980)Gene 8:279-300].

3.6.2. Construction of pSOD/env5b

Plasmid pSOD/env5b is a pBR322 derivative which contains the sequencescoding for env-5b [Sanchez-Pescador et al. (1985) supra] undertranscriptional control of a hybrid tac promoter. De Boer et al. (1983)supra.

Plasmid pSOD/env5b (FIG. 25) was constructed by cloning a 322 base pairssynthetic DNA fragment coding for env-5b into plasmid pSODCF2 [Steimeret al. (1986) J. Virol. 58:9-16], a vector containing the human SOD geneunder the control of the tacI promoter. Plasmid pSODCF2 is derived fromvector ptac5 [Hallewell et al. (1985) Nucl. Acid. Res. 13:2017] a pBR322derivative which contains the tac promoter, Shine Delgarno sequences anda polylinker as a substitution of the original pBR322 sequencescomprised between the EcoRI and PvuII restriction sites.

The 322 base pairs synthetic DNA fragment codes for the env-5b fragment(amino acid residues 557 to 677 as numbered in FIG. 5) with anadditional methionine to allow for translational initiation site. Thesynthetic gene was prepared by synthesizing oligonucleotides varying inlength between 34 and 48 bases, and purifying and kinasing themindividually according to standard procedures. Warner et al. (1984) DNA3:401. The solution was phenol extracted and ethanol precipitated. Thepellet was redissolved in 30 μl of a buffer containing 20 mM Tris-HCl(pH 8.0), 10 mM MgCl₂, and 10 mM dithiothreitol, and heated to 90° C.After allowing to cool over 3 hours, the solution was made 3 mM in ATP,brought up to a volume of 45 μl, and 5 μl of T4 DNA ligase (4×10⁵ U/mlBiolabs) added. The ligation was stopped after 10 minutes at 25° C. byphenol extraction and ethanol precipitation. The pellet was redissolvedin 80 μl H₂O in 10 μl of 10× high salt restriction buffer and digestedfor 2 hours at 37° C. with 5 μl each of NcoI and SalI (Biolabs). Thefull-length gene was then purified on a 7% native polyacrylamide gel,electroeluted, and cloned into NcoI/SalI-digested pBS-100.

The cloned env-5b gene was isolated as an NcoI/SalI fragment from theresulting plasmid and cloned into NcoI/SalI-digested pSODCF2 to giveplasmid pSOD/env5b. The resulting bacterial expression vector was usedto express the SOD/env-5b fusion protein in E. coli under the control ofthe tac-1 promoter. Hallewell et al. (1985) Nucleic Acids Res. 13:2017.

FIG. 15 shows the nucleotide sequence of the SOD/env-5b insert cloned inpSOD/env5b and the amino acid sequences derived for it. Sequences codingfor human SOD were derived from a human cDNA isolated as described inHallewell et al. (1985), supra. Sequences coding for env-5b werechemically synthesized by the phosphoramidite method as originallydescribed by Beaucage & Caruthers, (1981) Tetrahedron Lett. 22:1859.

3.6.3. Expression of SOD/env-5b Fusion Protein

E. coli D1210 cells were made competent for transformation following astandard procedure and transformed with pSOD/env5b. The transformationmix was plated on agar plates made with L-broth containing 100 μg/mlampicillin. Plates were incubated for 12 hours at 37° C.

Single ampicillin resistant colonies were transferred into L-brothcontaining 100 μg/ml ampicillin and grown at 37° C. Expression of env-5bwas induced by adding 100 mM IPTG to a final concentration of 1 mMfollowed by incubation at 37° C. for 2 hours.

Cells from induced cultures were pelleted and resuspended in Laemmlisample buffer. Laemmli (1970) Nature 227:680. After 3 cycles of boilingand freezing, portions of resultant lysates were analyzed on standarddenaturing acylamide gels. Laemmli (1970), supra. Proteins werevisualized by staining with Coomassie blue.

The extent of expression was initially determined by appearance of newprotein bands for induced candidate samples compared with controls.Proteins of molecular weights expected for the cloned DNA weredetermined by visual inspection of the gel lanes with reference to astandard protein band of known amount.

Authenticity of the expressed proteins was determined by standardwestern transfer of proteins to nitrocellulose and analysis withappropriate human or rabbit immune sera or mouse monoclonal antibodiesor by ELISA assays of soluble E. coli proteins using human immune serafrom AIDS patients.

3.6.4. Protein Purification

Single colonies of D1210 (pSOD/env5b) were inoculated into 2 ml aliquotsof L-broth containing 100 μg/ml ampicillin and the cultures were grownovernight at 37° C. The culture was made 15% glycerol by adding ¼ volumeof 75% sterile glycerol. The glycerol cell stocks aliquoted and quicklyfrozen in liquid nitrogen. The aliquots were stored at −70° C.

A frozen aliquot of the seed stock (200 μl) was thawed and used toinoculate 110 ml of L-broth with 0.01% ampicillin medium. The culturewas grown to saturation at 37° C. with agitation. A 15 ml portion of theculture was added to each of six (6) flasks containing 1.5 L of L-brothwith 0.01% ampicillin medium. The cultures were grown at 37° C. withagitation. During late log phase (after approximately 6 h.), env-5bexpression was induced by addition of IPTG to a final concentration of 1mM. Incubation was continued for an additional 2-4 h. at 37° C.

After the cells had completed the growth period they were harvested bycentrifugation and pooled. The packed cells were stored at either 4° C.or −20° C. The frozen (or refrigerated) cells were thawed and suspendedin 2 volumes of cell lysis solution (1.0 mM PMSF, 0.18 mM EDTA, 230 mMtris-HCl, 0.425 mg/ml Lysozyme) on ice for 1 h. Approximately 1.5volumes of DNA Digestion Solution (1.0 mM PMSF, 30 units/ml DNAse I, 1.0mM MgCl₂) was added and then the cell lysate was sonicated at roomtemperature for 30 minutes. The lysate was centrifuged at 113,000×g for15 minutes.

The supernatant was discarded and the pellet was suspended insolubilization buffer (0.1% β-mercaptoethanol in 7 M guanidine-HCl) androcked for 3 h. at 2-8° C. The solution was subjected toultracentrifugation at 25,500×g for 4 h. at 2-8° C. The pellet wasdiscarded, and the supernatant was diluted with 6 volumes of water, andthen incubated at room temperature for 1-2 h. The diluted supernatantwas centrifuged at 11,300×g for 30 minutes. Following centrifugation,the supernatant was discarded and the pellet was resuspended in 10-15volumes of elution and resuspension buffer (0.1% β-mercaptoethanol, 50mM Tris-HCl in 3.5 M guanidine-HCl, pH 7.4). The suspension wascentrifuged at 2,000×g for 10 minutes at 2-8° C. to remove any insolublematerial.

The resolubilized fraction was chromatographed on a Sephacryl S-300column and eluted with Elution and Resuspension Buffer. Fractions werecollected and the absorbance was monitored at 280 nm. Fractionscontaining env-5b as determined by SDS-PAGE were pooled. The pooledfractions were concentrated to ⅕ the original volume by ultrafiltrationin an Amicon unit under N₂ pressure.

The concentrate was dialyzed (MW cutoff 2000) against 50 to 100 volumesof urea-containing dialysis buffer (0.1% B-Mercaptoethanol, 50 mMTris-HCl in 7.5 M urea, pH 7.4) for 16-24 h. at 2-8° C. The proteinconcentration of the dialyzed env-5b was adjusted to 1.02.0 mg/ml (basedon modified Lowry assay).

3.7. Expression of β-gal-env Fusion Proteins.

3.7.1. Host-Vector System

A partial env protein is synthesized by E. coli D1210 transformed withplasmid pII-3. Plasmid pII-3 (ATCC No. 67549) is a bacterial expressionvector which contains the sequence for ⅔ (carboxyl end) of the envprotein fused to E. coli b-galactosidase DNA in the vector pNL291.Expression of the β-gal-env fusion protein is induced with IPTG.

3.7.2. Construction of pII-3

Plasmid pII-3 was constructed by cloning a 1855 bp BglII-XhoI fragmentcoding for ⅔ of the env protein. The fragment extends from nt 6604 to nt8460 (FIG. 5) and codes for env amino acid residues from number 276 tothe end of the env protein.

To prepare the plasmid, the 1856 bp fragment was isolated by gelelectrophoresis. The BglII-XhoI fragment was cloned into pNL291 whichhad been previously digested with BamHI and XhoI. Plasmid pNL291 is aderivative pUR291 [Ruther et al. (1983) EMBO J. 2:1791-1794] in which apolylinker containing the additional restriction sites NcoI, PvuII andXhoI was substituted between the BamHI and SalI sites. Plasmid pUR291 isan inducible E. coli expression vector which produces β-galactosidaseC-terminal fusion proteins.

3.7.3. Expression and Characterization of Fusion Protein

E. coli D1210 cells were transformed with 25-50 ng of pII-3. Thetransformation mix was plated onto L-Broth agar plates containing 100μg/ml ampicillin. Single amp^(R) colonies were grown in L-Broth/amp andexpression of the fusion protein was induced by IPTG addition (1 mM)followed by incubation. Cell extracts were prepared and analyzed by SDSgel electrophoresis and immunoassays using human immune sera or rabbitimmune sera.

Results of this analysis showed that a large molecular weight protein isinduced with IPTG. Expression levels are about 20 mg/l. This fusionprotein reacted with human immune sera from AIDS patients in ELISAassays.

4. Expression of HIV Polypeptides in Yeast

4.1. Expression of envH Peptide

4.1.1. Host-Vector System

A partial env protein is synthesized by S. cerevisiae 2150-2-3transformed with plasmid pDPC303. Plasmid pDPC303 is a yeast expressionvector which contains the sequence coding for ⅔ of the env protein,envH, as well as pBR322 sequences including the amp^(R) gene and2-micron sequences including the yeast leu 2-04 gene. Expression of envHis under regulation of the yeast pyruvate kinase promoter and terminatorsequences. east strain S. cerevisiae 2150-2-3 has the followinggenotype: Mat a, ade 1, leu 2-112, cir°. This strain was obtained fromDr. Leland Hartwell, University of Washington.

4.1.2. Construction of pDPC303

Plasmid pDPC303 contains an “expression cassette” (described below) forenvH cloned into the BamHI site of vector pCl/1. Vector pCl/1 containspBR322 and 2 micron sequences including the amp^(R) and yeast leu 2-04markers. It was derived from pJDB219d [Beggs (1978) Nature 275:104] byreplacing the pMB9 region with pBR322 sequences.

The “expression cassette” for envH consists of the following sequencesfused together in this order (5′ to 3′): yeast pyruvate kinase (PYK)promoter, envH coding region and PYK terminator. The PYK promoter andterminator regions were derived from the PYK gene isolated as describedin Burke et al. (1983) J. Biol. Chem. 258:2193-2201.

The envH fragment cloned into the expression cassette was derived fromARV-2 DNA and comprises a 1395 bp fragment which codes for env aminoacid residues 27 through 491 coded by nt 5857 to nt 7242 (FIG. 5). Inaddition, there are 5 extra codons fused in reading frame in the 5′ end,the first codon corresponding to a methionine, and 4 extra codons fusedin reading frame at the 3′ end followed by a stop codon. The extracodons were incorporated to facilitate cloning procedures exclusively.

FIG. 11 shows the coding strand of the nucleotide sequence cloned inpDPC303 and the amino acid sequence derived from it. DNA sequences thatare not underlined in the figure were derived directly from the ARV-2λ9B DNA described above. All other sequences were either chemicallysynthesized, or derived from the PYK vector.

4.1.3. Transformation and Expression

Yeast cells S. cerevisiae 2150-2-3 (Mat a, ade1, leu 2-04, cir°) weretransformed as described by Hinnen et al. (1978) Proc. Natl. Acad. Sci.USA 75:1929-1933, and plated onto leu⁻ selective plates. Single colonieswere inoculated into leu⁻ selective media and grown to saturation. Cellswere harvested and the env protein was purified and characterized asdescribed below.

4.1.4. Purification of envH Protein

4.1.4.1. Cell Breakage

Frozen S. cerevisiae 2150-2-3 (pDPC303) are thawed and suspended in 1volume of lysis buffer (1 μg/ml pepstatin, 0.001 M PMSF, 0.001 M EDTA,0.15 M NaCl, 0.05 M Tris-HCl pH 8.0), and 1 volume of acid-washed glassbeads are added. Cells are broken in a noncontinuous system using a 300ml glass unit of Dyno-mill at 3000 rpm for 10 min. The jacket is keptcool by a −20° C. ethylene glycol solution. Glass beads are decanted byletting the mixture set for 3 minutes on ice. The cell extract isrecovered and centrifuged at 18,000 rpm (39,200×g) for 35 min. Thesupernatant is discarded and the precipitate (pellet 1) is furthertreated as indicated below.

4.1.4.2. SDS Extraction of Insoluble Material

Pellet 1 is resuspended in 4 volumes of Tris-HCl buffer (0.01 MTris-HCl, pH 8.0, 0.01 M NaCl, 0.001 M PMSF, 1 μg/ml pepstatin, 0.001 MEDTA, 0.1% SDS) and extracted for 2 h at 4° C. with agitation. Thesolution is centrifuged at 6,300×g for 15 min. The insoluble fraction(pellet 2) is resuspended in 4 volumes (360 ml) of PBS (per liter: 0.2 gKCl, 0.2 g KH₂PO₄, 8.0 g NaCl, 2.9 g Na₂HPO₄.12H₂O), 0.1% SDS, 0.001 MEDTA, 0.001 M PMSF, 1 μg/ml pepstatin, and centrifuged at 6,300×g for 15min. The pellet (pellet 3) is suspended in 4 volumes of PBS, 0.2% SDS,0.001 M EDTA, 0.001 M PMSF, 1 μg/ml pepstatin and is extracted for 12 hat 4° C. with agitation on a tube rocker. The solution is centrifuged at6,300×g for 15 min. The soluble fraction is recovered for furtherpurification as indicated below. (The pellet can be reextracted byresuspending it in 4 volumes of 2.3% SDS, 5% β-mercaptoethanol, andboiling for 5 min. After boiling, the solution is centrifuged at 6,300×gfor 15 min. The soluble fraction is recovered for further purification.)

4.1.4.3. Selective Precipitation and Gel Filtration

The soluble fraction is concentrated by precipitation with 30% ammoniumsulfate at 4° C. The pellet (pellet 4) is resuspended in 2.3% SDS, 5%β-mercaptoethanol, and chromatographed on an ACA 34 (LKB Products) gelfiltration column. The column is equilibrated with PBS, 0.1%. SDS, atroom temperature. Chromatography is developed in the same solution witha flow rate of 0.3 ml/min. Five ml fractions are collected, pooled andcharacterized by protein gel electrophoresis, Western analysis, andELISA. If needed, pooled fractions are concentrated by vacuum dialysison Spectrapor #2 (MW cutoff 12-14K).

4.1.5. Characterization of Recombinant envH

SDS polyacrylamide gel analysis (12% acrylamide gels) showed that a new55,000 dalton protein was being synthesized in yeast cells transformedwith the env-containing vector. The 55,000 dalton protein is absent fromcells transformed with control plasmid (vector without env insert). Theidentity of env was confirmed by both ELISA (Section 3.1.6.3.) andWestern analysis using AIDS patient serum. In both assays the 55,000dalton protein showed immunoreactivity. No reactivity was obtained withserum from a normal individual.

4.2. Expression of Env Subregion Polypeptides

The following example describes the expression of DNA coding regionsdefined as env-1, env-2, env-3, env-4 and env-5b (see FIG. 13). Env-1and env-4 approximate the N- and C-terminal halves of gp120env,respectively. Env-2 and env-3 approximate the entire env polypeptidemoieties of gp120 and gp41, respectively. Env-5b corresponds to theregion of gp41env thought to be external to the cell membrane.

4.2.1. Env-1

4.2.1.1. GAP Promoter

Proviral ARV-2 sequences were isolated from phage λARV-2 (7D) andsubcloned into plasmid pUC19. Yanisch et al. (1985) Gene 33:103-119. Theplasmid containing the proviral sequences was named pUC19ARV7D/7. Toisolate the env-1 region, the plasmid was cut with FokI at cys27 of theenv region (nt5857, FIG. 5) and with BglII at arg276 (nt6604, FIG. 5).This provided a nucleotide segment with a coding capacity for 28 kD fromthe N-terminus of gp120 without the 29 amino acid signal sequence of theenv gene product. A synthetic, NcoI/FokI adaptor, with the followingsequence, was ligated to the env-1 segment:

5′-CATGGCTATC        CGATAGACAT-5′A second adaptor, for BglII/SalI, was also ligated to the env-1 segment.This second adaptor had the following sequence:

5′-GATCTTGATAGG        AACTATCCAGCT-5′The first synthetic adaptor contains an in-frame initiation codon, andthe second synthetic adaptor contains an in-frame stop codon.

The env-1 segment modified with the synthetic adaptors was then ligatedinto pPGAP1 previously linearized with NcoI and SalI. Plasmid pPGAP1 hasbeen previously described. EPO Pub. No. 164,556. It contains a yeastglyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter and terminatorsequences flanking NcoI and SalI cloning sites. The ligation of themodified env-1 segment into pPGAP1 produces plasmid pPGAP/FGenv, whichcontains the env-1 sequence fused directly to the GAPDH promoter andterminator sequences. The expression cassette containing GAPDHpromoter-env-1-GAPDH terminator was excised by digestion of pPGAP/FGenvwith BamHI and gel purification of the fragment. The expression cassettewas cloned into BamHI-digested pCl/1 (see Section 4.1.2) to yieldpCl/1FGenv.

Plasmid pCl/1FGenv was used to transform yeast strain AB110 (Matα, ura3-52, leu 2-04 or both leu 2-3 and leu 2-112, pep 4-3, his 4-580, cir°;see EPO Pub. No. 620,662 & Section 4.5.2) as described previously.Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA 75:1929. Yeast coloniestransformed with the expression plasmid were grown in synthetic completemedia lacking leucine at a concentration of 2% glucose. Large-scalecultures were grown in YEP medium with 2% glucose as described inSection 4.1.3. Lysates were prepared from yeast cultures as describedpreviously, and were then separated into soluble and insoluble fractionsby centrifugation, and analyzed by polyacrylamide gel electrophoresis,see Section 4.1.4. A heavily expressed protein corresponding to env-1was readily discernible in the insoluble fraction by Coomassie bluestaining. This protein also migrated at a molecular weight ofapproximately 28 kD, as predicted from the DNA sequence.

4.2.2. Env-2

The env-2 polypeptide is similar to the previously described envH(Section 4.1.1) and corresponds to the viral gp120 glycoprotein. Env-2differs from envH in the 5 amino terminal residues and is under theregulatory control of the GAPDH promoter as described for the expressionof env-1. Env-2 is a polypeptide having the amino acid sequence ofgp120env residues 26510.

4.2.2.1. Construction of pAB24-GAP-env2

Plasmid pAB24-GAP-env2 (FIG. 28) contains an “expression cassette” forthe env gene cloned into the BamHI site of the yeast shuttle vectorpAB24 (below). Expression of the env gene is under regulatory control ofthe GAPDH promoter and the PYK terminator. Construction ofpAB24-GAP-env2 was accomplished by ligating (i) an approximately 952 bpBamHI-StuI fragment from plasmid pCl/1-FGenv (Section 4.2.1) whichcontains the GAPDH promoter and the envelope sequences coding for aminoacids 26-267, and (ii) an approximately 1474 bp StuI-BamHI fragment fromplasmid pDPC302 (which is similar to pDPC303 in Section 4.1.1. exceptthat it extends 57 nucleotides in the 3′ direction of the envelopecoding region), which codes for env amino acids 267-510 and the PYKterminator, into plasmid pAB24 which had been previously digested withBamHI and treated with alkaline phosphatase. The direction oftranscription in the “expression cassette” is in the opposite directionof the tetracycline gene of the pBR322 sequences.

Plasmid pAB24 (FIG. 27) is a yeast shuttle vector which contains thecomplete 2μ sequence [Broach, in: Molecular Biology of the YeastSaccharomyces, Vol. 1, p. 445, Cold Spring Harbor Press (1981)] andpBR322 sequences. It also contains the yeast URA3 gene derived fromplasmid YEp24 [Botstein et al. (1979) Gene 8:17] and the yeast LEU^(2d).gene derived from plasmid pCl/1. EPO Pub. No. 116,201. Plasmid pAB24 wasconstructed by digesting YEp24 with EcoRI and religating the vector toremove the partial 2μ sequences. The resulting plasmid, YEp24ΔRI, waslinearized by digestion with ClaI and ligated with the complete 2μplasmid which had been linearized with ClaI. The resulting plasmid,pCBou, was then digested with XbaI and the 8605 bp vector fragment wasgel isolated. This isolated XbaI fragment was ligated with a 4460 bpXbaI fragment containing the LEU²d gene isolated from pCl/1; theorientation of the LEU²d gene is in the same direction as the URA3 gene.Insertion of the expression cassette was in the unique BamHI site of thepBR322 sequences, this interrupting the gene for bacterial resistance totetracycline.

4.2.2.2. Transformation and Expression

Yeast cells S. cerevisiae 2150-2-3 (Mat a, ade1, leu 2-04, cir°) weretransformed as described by Hinnen et al; [(1978) Proc. Natl. Acad. Sci.USA 75:1929-1933] and plated onto leu-selective plates. Single colonieswere inoculated into leu-selective media and grown to saturation. Theculture was further inoculated into YEP 2% glucose media. Cells wereharvested and the env-2 protein was purified and characterized asdescribed below.

4.2.2.3. Purification of Env-2 Protein

Frozen S. cerevisiae 2150-2-3 (pAB24-GAP-env2) were thawed and suspendedin 1 volume of lysis buffer (1 μg/ml pepstatin, 0.001 M PMSF, 0.001 MEDTA, 0.15 M NaCl, 0.05 M Tris-HCl pH 8.0), and 1 volume of acid-washedglass beads are added. Cells are broken in a non-continuous system usinga 300 ml glass unit of Dyno-mill at 3000 rpm for 10-25 min. The jacketis kept cool by a −20° C. ethylene glycol solution. Class beads aredecanted by letting the mixture set for approximately 3 minutes on ice.The cell extract is recovered and centrifuged at 18,000 rpm (39,200×g)for 35 min. The supernatant is discarded and the precipitate (pellet 1)is further treated as indicated below.

Pellet 1 is resuspended in 4 volumes of Tris-HCl buffer (0.01 MTris-HCl, pH 8.0, 0.01 M NaCl, 0.001 M PMSF, 1 μg/ml pepstatin, 0.001 MEDTA, 0.1% SDS) and extracted for 2 h at 4° C. with agitation. Thesolution is centrifuged at 6,300×g for 15 min. The insoluble fraction(pellet 2) is resuspended in 4 volumes of PBS (per liter: 0.2 g KCl, 0.2g KH₂PO₄, 8.0 g NaCl, 2.9 g Na₂HPO₄.12H₂O), 0.1% SDS, 0.001 M EDTA,0.001 M PMSF, 1 mg/ml pepstatin, and centrifuged at 6,300×g for 15 min.The pellet (pellet 3) is suspended in 4 volumes of PBS, 0.2% SDS, 0.001M EDTA, 0.001 M PMSF, 1 μg/ml pepstatin and is extracted for 12±3 h at4° C. with agitation on a tube rocker. The solution is centrifuged at6,300×g for 15 min. The soluble fraction is recovered for furtherpurification as indicated below. (The pellet can be reextracted byresuspending it in 4 volumes of 2.3% SDS, 5% B-mercaptoethanol, andboiling for 5 min. After boiling, the solution is centrifuged at 6,300×gfor 15 min. The soluble fraction is recovered for further purification.)

The soluble fraction is concentrated by precipitation with 30% ammoniumsulfate at 4° C. The pellet (pellet 4) is resuspended in 2.3% SDS, 5%β-mercaptoethanol, and chromatographed on an ACA 34 (LKB Products) gelfiltration column. The column is equilibrated with PBS, 0.1% SDS, atroom temperature. Chromatography is developed in the same solution witha flow rate of 0.3±0.1 ml/min. 5±2 ml fractions are collected, pooledand characterized by protein gel electrophoresis, Western analysis, andELISA. If needed, pooled fractions are concentrated by vacuum dialysison Spectrapor #2 (MW cutoff 12-14K), or by membrane filtration using anAmicon concentrator using a PM10 membrane.

SDS polyacrylamide gel analysis (12% acrylamide gels) showed that a new55,000 dalton protein was being synthesized in yeast cells transformedwith the env-containing vector. The 55,000 dalton protein is absent fromcells transformed with control plasmid (vector without env insert). Theidentity of env-2 was confirmed by both ELISA and Western analysis usingAIDS patient serum. In both assays the 55,000 dalton protein showedimmunoreactivity. No reactivity was obtained with serum from a pool ofnormal individuals.

4.2.2.4. Immunogenicity

To determine the immunogenicity of polypeptides expressed in yeast,purified env-2 (Section 4.2.2.3) was used to immunize rabbits. Rabbitsreceived perilymphnodal injections of 250 μg of purified env-2polypeptide in complete Freund's adjuvant. Twenty-one days later, therabbits were boosted with intramuscular injections of 250 μg of env-2polypeptide in incomplete Freund's adjuvant. Ten days later the animalswere bled and then set up on a schedule of boosting, bleeding 10 dayslater, and reboosting after 21 days. Antibody titers to env-2polypeptide for the rabbits were measured using ELISA. Antibodies fromthe rabbits reacted specifically with env glycoproteins in virus (gp120)and in infected cells (gp160). Thus, the polypeptide moiety representingthe N-terminal domain of the env region is immunogenic when expressed ina heterologous host species.

4.2.3. Env-3

4.2.3.1. GAP Promoter

Env-3, the gp41env equivalent (AA 529-855 of env), was prepared bycutting the env insert of plasmid pUC19ARV7D/7 (Section 4.2.1.1) at theHgaI site (nt7401, FIG. 5) and at the XhoI site 3′ to the envtermination codon (nt8460, FIG. 5). The resulting env-3 segment wasmodified by the addition of synthetic adapters. The 5′ end was modifiedby the addition of an NcoI/HgaI adapter which reintroduced the codingsequence to met529 (nt7817, FIG. 5). The linker had the followingsequence:

5′-CATGGGCGCCGTTTCTTTGACCTTGACC-3′    3′-CCGCGGCAAAGAAACTGGAACTGGCATGT-5′A second synthetic XhoI/SalI adapter molecule was prepared and ligatedto the XhoI end of env-3, the adapter having the following sequence:

5′-TCGACCTCGAGG-3′     3′-GGAGCTCCAGCT-5′The HgaI/XhoI fragment was cloned together with the above linkers intoNcoI/SalI-digested pPGAP/FGenv (Section 4.2.1.1), and the resultingplasmid, pPGAP/HXenv, was digested with BamHI. The BamHI expressioncassette was cloned into pCl/1. The resulting expression vectorpCl/1GAP/HXenv was expressed as described above after transformation ofyeast strain AB110 (Section 4.2.1). Extreme toxicity, as evidenced byslow growth of cells, was observed when this gene was expressedconstitutively in yeast under control of the GAPDH promoter.

4.2.3.2. ADH-2/GAPDH Promoter

In order to express env-3 under the control of the glucose-regulableADH-2/GAPDH (or ADH-2/GAP) promoter, the Nco-I/BamHI fragment containingthe env-3 coding region and the GAPDH terminator was excised frompPGAP/HXenv. This was cloned together with the ADH-2/GAPDH promoter as aBamHI/Nco-I fragment (from pJS103) into BamHI-digested and phosphatizedpCl/1. The resulting expression vector, pCl/1ADH-2/GAP/HXenv, wasexpressed as described below after transformation of yeast strain AB110.

Plasmid pJS103, which contains the hybrid ADH-2/GAPDH promoter employedabove, was constructed as follows. The ADH-2 portion of the promoter wasconstructed by cutting a plasmid containing the wild-type ADH2 gene fromplasmid pADR2 [Beier et al. (1982) Nature 360:724-728] with restrictionenzyme EcoR5, which cuts at position +66 relative to the ATG startcodon, as well as in two other sites in pADR2, outside of the ADH2region. The resulting mixture of a vector fragment and two smallerfragments was resected with Bal31 exonuclease to remove about 300 bp.Synthetic XhoI linkers were ligated onto the Bal31-treated DNA. Theresulting DNA linker vector fragment (about 5 kb) was separated from thelinkers by column chromatography, cut with restriction enzyme XhoI,religated, and used to transform E. coli to ampicillin resistance. Thepositions of the XhoI linker were determined by DNA sequencing. Oneplasmid which contained an XhoI linker within the 5′ nontranscribedregion of the ADH2 gene (position −232 from ATG) was cut with therestriction enzyme XhoI, treated with nuclease S1, and subsequentlytreated with the restriction enzyme EcoRI to create a linear vectormolecule having 1 blunt end at the site of the XhoI linker and an EcoRIend. The GAP portion of the promoter was constructed by cutting plasmidpPGAP with the enzymes BamHI and EcoRI, followed by the isolation of the0.4 Kbp DNA fragment. This purified fragment was then completelydigested with the enzyme AluI and an approximately 200 bp fragment wasisolated. This GAP promoter fragment was ligated to the ADH-2 fragmentpresent on the linear vector described above to give plasmid pJS103.

S. cerevisiae AB110 was transformed with the ADH-2/GAPDH constructions,and the cultures grown initially in synthetic complete media lackingleucine with 8% glucose. Env-3 was induced by diluting the culture 1:25into YEP with 1% glucose and allowing growth at 30° C. for 24 hours.Normal cell growth was observed; however, complex expression productswere observed. In an immunoblot assay with AIDS sera, the proteinappeared as a multiplet of immunoreactive bands, including a major bandat about 37 kD and five additional bands of increasing molecular weight.As demonstrated by treatment with endoglycosidase-H prior to gel andimmunoblot analysis, these additional bands were due to glycosylation.Inspection of the gp41 DNA sequence shows five potential N-linkedcarbohydrate addition sites. Since env-3 encodes a polypeptide with acalculated molecular weight of 40.5 kD, the gel mobility of env-3 ataround 37 kD may indicate either aberrant electrophoresis properties, orC-terminal processing analogous to that proposed for infectedT-cell-derived gp41.

4.2.4. Env-4

The env-4 region approximates the carboxy-terminal half of the gp120glycoprotein from the envelope gene and corresponds to amino acidsGlu-272 to Arg-509 (FIG. 5). Env-4 also contains a methionine at theN-terminus which serves as an initiation codon for the yeast expressionsystem.

Expression of the env-4 protein was initially attempted as a directexpression product in yeast which failed to provide any detectableproduct. Successful expression of the env-4 protein was achieved as anSOD fusion product in yeast.

4.2.4.1. pBS24/SF2env4/GAP

The 3′ end of the env-4 coding sequence was modified by M13 mutagenesisto generate 2 stop codons in frame after Arg-509, and by addingrestriction sites for HindIII and SalI. Plasmid pSV-7c/env (Section2.1.2) was digested with HindIII and XhoI and an approximate 2830 bpfragment was gel isolated. The fragment was cloned into M13-mp19 andsingle stranded template was generated. M13 mutagenesis was performedusing the following primer:

5′-GAACATAGCTGTCGACAAGCTTCATCATCTTTTTTCT-3′.

A single plaque designated M13Fenv3-447 was isolated and confirmed byM13 sequencing to contain the inserted stop codons and new restrictionsites for HindIII and SalI. M13 replicative-form DNA was prepared forM13Fenv3-447 by standard methods, and an approximately 713 bp BglII(position 6604, FIG. 5) to SalI fragment was excised and gel purified.This fragment was ligated to the following NcoI-BglII linker, whichcodes for a methionine initiation codon and the first four amino acidsof the env-4 protein:

    MetGluValValIleArg 5′-CATGGAGGTAGTAATTA-3′        CTCCATCATTAATCTAGand then cloned into pPGAP1 [EPO Pub. No. 164,556] which was previouslydigested with NcoI and SalI and gel isolated. The approximate 1130 bpBamHI-SalI fragment containing the GAPDH promoter and the env-4 gene wasexcised and cloned into pBS24 (below), which was previously digestedwith BamHI and SalI and gel isolated, to give plasmid pBS24/SF2env4/GAP.

Plasmid pBS24/SF2env4/GAP was transformed into S. cerevisiae strains2150-2-3 and AB110 as described previously. Cultures from singlecolonies were grown and analyzed for expression by SDS polyacrylamidegel electrophoresis. No env-4 protein was detected by either Coomassiestained gels or western blot analysis.

4.2.4.2. pBS24

Plasmid pBS24 is a derivative of pAB24 as described in Section 4.2.2.1.Plasmid pAB24 was digested with BamHI and SalI (which cut within thetetracycline gene of the pBR322 sequences) and gel purified. The vectorwas then ligated with a synthetic adapter of the following sequencewhich created new unique BglII and BamHI sites:

       BglII           BamHI 5′-GATCAGATCTAAATTTCCCGGATCC-3′       TCTAGATTTAAAGGGCCTAGGAGCT (BamHI)                      (SalI)The resulting vector, pAB24ΔBL was then digested with BamHI and BglIIand gel purified. The linearized vector was ligated with the BamHIcassette excised and purified from pSOD/env-5b (Section 4.2.5) to givepBS24. The cassette contains the hybrid ADH-2/GAPDH promoter andα-factor terminator with an NcoI-SalI insert of the SOD/env-5b fusiongene. The cassette is oriented in pBS24 such that the direction oftranscription from the ADH-2/GAPDH promoter is in the opposite directionto that of the inactivated tetracycline gene of the pBR322 sequences.

4.2.4.3. pBS24/SOD-SF2env4

Since there was no detectable expression of env-4 from the directexpression system, an SOD/env-4 fusion gene was constructed. PlasmidpBS24/SF2env4/GAP was digested with NcoI and SalI and the approximate713. bp env-4 gene was gel isolated. The env-4 gene was ligated intopSODCF-2 (Section 3.5) which was previously digested with NcoI and SalIand gel purified, to give pCF2-SODenv4. Plasmid pSODCF2 is a bacterialexpression vector which allows for the C-terminal fusions ofheterologous genes with the human SOD gene and is under the control ofthe tacl promoter. When plasmid pCF2SODenv4 was transformed into E. colistrains D1210 and RR1ΔM15 and analyzed for expression as describedpreviously (Sections 3.4.4 and 3.6), no expression of SOD/env-4 fusionprotein was detected. Plasmid pCF2SODenv4 was digested with StuI andSalI to isolate the 3′ half of the SOD gene fused with the env-4 gene.This fragment was ligated to pSI8 which had been previously digestedwith StuI and SalI and gel isolated to give plasmid pSI8—SODenv4.Plasmid pSI8 (Section 4.2.5) contains the hybrid ADH-2/GAPDH promoterand the α-factor terminator sequences flanking the SOD-insulin fusiongene. The resulting plasmid pSI8—SOD/env-4 was digested with BamHI andSalI to excise a fragment containing the ADH-2/GAPDH promoter andSOD/env-4 fusion gene. This fragment was gel isolated and cloned intopBS24 which had been previously digested with BamHI and SalI and gelisolated to give pBS24/SOD-SF2env4 (FIG. 26).

4.2.4.4. Transformation and Expression.

The plasmid pBS24/SOD-SF2env4 was transformed into yeast cells S.cerevisiae 2150-2-3 and S. cerevisiae AB116 (mat a, leu 2, trp 1, ura3-58, pro 1-1122 (prot. B), pep 4-3 (prot. A), pre 1-407 (prot. C),cir°) as described previously [Hinnen et al. (1978) Proc. Natl. Acad.Sci. USA 75:1929-1933] and plated onto Leu sorbitol plates. Strain AB116was isolated by curing S. cerevisiae strain BJ2168 of its 2 micronplasmid by standard methods. BJ2168 is available from the Yeast GeneticStock Center, Department of Biophysics and Medical Physics, Universityof California, Berkeley 94720.

Cultures were grown as follows: a loopfull of cells from the individualcolonies were inoculated into 3 ml of Leu⁻, 8% glucose media andincubated in an air shaker at 30° C. for 16-18 h. The 3 ml culture wastransferred to 40 ml of fresh Leu⁻, 8% glucose media and incubated in anair shaker at 30° for 24 h. The 40 ml culture was transferred to 1 literof YEP, 1% glucose media (or YEP, 2.5% Ethanol media for 2150-2-3 cells)and incubated in an air shaker at 30° for an additional 48 h. The cellswere harvested by centrifugation and stored at −20° C.

Expression of the SOD/env-4 fusion protein was analyzed by bothpolyacrylamide gel electrophoresis and western blot. Cells are disruptedby glass bead lysis method in 0.1 M NaPo₄ (pH 7.4), 0.1% Triton lysisbuffer. After lysis the soluble and insoluble fractions are separated bycentrifugation. The insoluble pellet is solubilized in Laemmli gelloading buffer and run on a 12.5% acrylamide SDS gel. Laemmli (1970)Nature 227:680. A band migrating at approximately 44 Kd molecular weight(expected size of SOD/env-4 fusion protein) was observed. This same bandalso reacted on a western blot with AIDS positive human sera but notwith control sera. Expression levels of SOD/env-4 fusion protein wereapproximately equal for the two different strains. Protein purificationwas carried out essentially as described in Section 4.2.2.3. for env-2.

4.2.5. ySOD/env-5b Fusion Protein

The region of the envelope gene corresponding to amino acids alanine-557through tryptophan-677 is termed env-5b as described in Section 3.6 andis also expressed as a stable fusion protein with hSOD in yeast. AStuI-SalI fragment containing most of the SOD gene fused to the env-5bgene was removed from pSOD/env5b (Section 3.6) and cloned into pSI8which had been previously digested with StuI and SalI and gel isolatedto give pSI8/SOD-env5b. Plasmid pSI8 (described below) contains theADH-2/GAPDH hybrid promoter and α-factor terminator sequences flankingthe SOD-insulin fusion gene. The resulting plasmid, designatedpSI8/SOD-env5b, was digested with BamHI and the fragment containing thepromoter-ySODenv-5b fusion-terminator was isolated and cloned into theBamHI cut and phosphatased pAB24 to give pYSOD/env-5b, which was used totransform the yeast strain 2150, as described above. Expression of theySOD/env-5b fusion protein was induced by diluting a starter cultureinto YEP containing 1% ethanol.

Lysates were isolated and prepared as described above. A heavilyexpressed protein corresponding to the SOD/env-5 fusion was readilydiscerned in the insoluble fraction by Coomassie blue staining. Thisprotein migrated at a molecular weight of approximately 30.6 kD, aspredicted from its DNA sequence. This fusion protein was alsosubsequently shown to have a high proportion of reactivity to AIDSpatients' sera.

Plasmid pSI8 is a derivative pYASI1, the latter being described incommonly owned U.S. patent application Ser. No. 845,737, filed on 28Mar. 1986 by Cousens et al. and EPO Pub. No. 196,056, the disclosures ofwhich are expressly incorporated herein by reference. Essentially, pSI8contains: The hybrid ADH-2/GAPDH promoter (as a 1.3 kb Bam-Nco fragment)derived from plasmid pJS104 (described below); an SOD-insulin fusiongene (as a 736 bp Nco-Sal fragment) derived from a derivative of pYSI1(U.S. Ser. No. 845,737); and the α-factor terminator isolated as a 277bp SalI-EcoRI fragment in which the EcoRI site has been filled in withKlenow fragment and BamHI linkers ligated to give a SalI-BamHI fragment[Brake et al. (1984) Proc. Natl. Acad. Sci. USA 81:4642-4646]; allcloned into a pBR322 derivative in which the segment between EcoRI andSalI is deleted and Bam linkers attached. Plasmid pJS104 is the same asplasmid pJS103 (Section 4.2.3.2), except that the GAPDH fragment of theADH-2/GAPDH promoter is about 400 bp, as opposed to 200 bp. Theconstruction of pJS104 was the same as pJS103, except that during thepreparation of the GAPDH portion of the promoter, the 0.4 KbpBamdI-EcoRI fragment was partially digested with AluI to create ablunt-end near the BamHI site.

4.2.6. Env 4-5

The env 4-5 polypeptide corresponds to the region of the envelope genewhich approximates the C-terminus of gp120 and the N-terminus of gp41,amino acids 272 to 673 (FIG. 5). Plasmid pBS24.1/SOD-SF2env4-5 was usedto express the env 4-5 polypeptide as an SOD fusion protein under theregulatory control of the ADH-2/GAPDH promoter in yeast.

4.2.6.1. Construction of pBS24.1/SOD-SF2env4-5

To construct plasmid pBS24.1/SOD-SF2env4-5 a 1.2 kbp BglII-SalI fragmentwhich corresponds to nucleotides 6603-7795 (see FIG. 5) was isolatedfrom pSV7dARV160T-tpa and ligated into pBS24/SOD-SF2env4 (see Section4.2.4.3.) which had previously been digested with BglII and SalI.Plasmid pSV7dARV160T-tpa is a plasmid which contains the HIV envelopegene to which two stop codons, a HindIII, and SalI site had beenintroduced at position 7798 by M13 mutagenesis with the followingmutagenic primer:

5′-CTTTATATACGTCGACAAGCTTCATCAGCTAAACCAA-3′

4.2.6.2. Transformation and Expression

The plasmid pBS24.1/SOD-SF2env 4-5 was transformed into yeast cells S.cerevisiae AB116 [mat a, leu 2, trp 1, ura 3-58, pro 1-1122 (prot. B),pep 4-3 (prot. A), pre 1-407 (prot. c), cir°] as described previously[Hinnen, et al. (1978) Proc. Natl. Acad. Sci. USA 75:1929-1933] andplated on Leu⁻ sorbitol plates. The cultures were grown and expressionanalyzed as previously described in Section 4.2.4.4. Expression levelsof the SOD/env4-5 fusion protein were approximately 15-20% of theinsoluble protein fraction as estimated by Coomassie-blue staining ofPAGE-treated samples.

4.2.6.3. Protein Purification

Cultures of AB116 (pBS24.1/SOD-SF2env4-5) were grown in 3 ml Leu ⁽⁻⁾, 8%glucose medium overnight at 30° C. The 3 ml was inoculated into 50 mlLeu⁻, 8% glucose medium for 24 h at 30° C. The 50 ml was then inoculatedinto 1 liter of YEP, 1% glucose and grown 30-48 h for induction ofprotein expression. The cells expressing SOD/env 4-5 were harvested andfurther processed as described for the purification of env-2 (Section4.2.2.3.).

4.3. p31pol

4.3.1. GAP/ADH2 Promoter

The ARV248NL fragment (Section 3.4.2.3) was cloned into pBS100previously cut with NcoI and SalI. pBS100 (below) is a bacterial vectorderived from pAB12 with a BamHI cassette consisting of the GAP-ADH2promoter (i.e., the ADH-2/GAPDH promoter), an ARV-env gene as anNcoI-SalI fragment, and the GAP terminator. The BamHI cassette from apositive clone of pBS100/p31/GAP-ADH2 was cloned into pAB24 (Section4.2.2.1), a yeast vector with both ura and leu selection capabilities.Both orientations of the cassette in this vector were screened for andused to transform the yeast strain AB110 (Mat a, ura 3-52, leu 2-04, orboth leu 2-3 and leu 2-112, pep 4-3, his 4-580, cir°). These cells wereplated in both ura⁻ and leu⁻ plates. Also, ura⁻ cells were plated ontoleu⁻ plates.

Three different induction procedures were done: (1). Ura⁻ coloniespatched on ura⁻ plates were induced for 24 h in YEP/1% glucose. Both aWestern and a polyacrylamide gel were run on these samples. Both resultswere negative. (2). Colonies from ura⁻ plates patched on leu⁻ plateswere induced in either leu⁻/3% ethanol or YEP/1% glucose for 24 h. AWestern and a polyacrylamide gel were run on these samples and theresults were also negative. (3). Colonies from leu⁻ plates patched onleu⁻ plates were induced in either leu⁻/3% ethanol or YEP/1% glucose for24 h. The polyacrylamide gel showed a negative result. No Western wasrun on these samples.

Plasmid pBS100 is a yeast expression cassette vector cloned into apBR322 derivative, pAB12. The expression cassette contains the hybridADH-2/GAPDH promoter and the GAPDH terminator flanking a gene segmentfrom the envelope gene. The ADH-2/GAPDH promoter is a 1200 bp BamHI-NcoIfragment isolated from pJS103 (Section 4.2.3.2) and the GAPDH terminatoris a 900 bp SalI-BamHI fragment isolated from plasmid pPGAP1 (Section4.2.1.1). Plasmid pBS100 also contains a non-essential fragment betweenthe NcoI and SalI sites which is replaced by gene fragments of interest.The expression cassette can be removed from pBS100 by digestion withBamHI and cloned into yeast shuttle vectors for introduction into yeastcells.

Plasmid pAB12 is a pBR322 derivative lacking the region between thesingle HindIII and SalI sites and containing a BamHI linker insertedbetween the unique EcoRI site. This vector was constructed by digestingpBR322 to completion with HindIII and SalI, followed by limiteddigestion with Bal31 nuclease, repair of the ends so created with theKlenow fragment of E. coli DNA polymerase I, and blunt-end ligation withT4 DNA ligase to reform closed covalent circles. The plasmid was thenopened up with EcoRI, treated with the Klenow fragment of E. coli DNApolymerase I (to fill-in the 5′ overhangs), blunt-end ligated with BamHIlinkers, digested with BamHI to remove excess linkers, and then ligatedto form closed circles.

4.3.2. GAP Promoter

The pBS100/p31/GAP-ADH2 plasmid was cut with BamHI and NcoI and thefragment containing the p31 gene (NcoI-SalI) and the GAP terminator(SalI-BamHI) was gel purified. pCl/1-alpha 1 antitrypsin/GAP was alsocut with NcoI and SalI and the fragment including the GAP promoter(NcoI-BamHI) and a portion of pCl/1 (BamHI-SalI) was gel isolated aswell. Both fragments were ligated with the yeast vector pCl/1 previouslycut with BamHI and SalI. The BamHI cassette can only be cloned in asingle orientation in this case. The resulting DNA was used to transformyeast strains AB110 and PO17 (Mat a, leu 2-04, cir°) and the cells wereplated on leu⁻ plates. The transformation using strain PO17 gave notransformants.

Colonies from leu⁻ plates were-grown in 3 ml of leu⁻/2% glucose for 24h. Yeast was analyzed on polyacrylamide gels stained by Coomassie Bluewith negative results. No Western was run on these samples.

4.3.3. SOD-p31 Fusion Protein

The construction of the p31pol expression vector is shown schematicallyin FIG. 23. The DNA and amino acid sequences of the SOD/p31 insert areshown in FIG. 24.

4.3.3.1. pCl/1-pSP31-GAP-ADH2

For the construction of a gene for a fused protein SOD-p31 to beexpressed in yeast, plasmid pSI4/392 was used. This plasmid contains theSOD gene fused to the proinsulin gene under the regulation of theADH-2/GAP promoter. The proinsulin gene is located between EcoRI andSalI restriction sites. To substitute the proinsulin gene with theARV248NL (Section 3.4.2.3) fragment, two oligomers designated ARV-300and ARV-301, respectively, were synthesized using phosphoramiditechemistry. The sequences generate cohesive ends for EcoRI and NcoI oneach side of the molecule when the two oligomers are annealed. ARV-300and ARV-301 have the sequences:

ARV-300  5′ AATTCAGGTGTTGGAGC                 GTCCACAACCTCGGTAC5′  ARV-301

Two micrograms of pSI4/39-2 linearized with EcoRI were ligated to 100picomoles each of phosphorylated ARV-300 and dephosphorylated ARV-301 inthe presence of ATP and T4 DNA ligase in a final volume of 35 μl. Thereaction was carried out at 14° C. for 18 h. The DNA was furtherdigested with SalI and the fragments were resolved on a 1% low meltingpoint agarose gel and a fragment containing the vector plus the SOD gene(˜6.5 kb) was purified as described above and resuspended in 50 μl ofTE. Five μl of this preparation were ligated to 5 μl of ARV248NL in 20μl final volume for 18 h at 14° C. and 5 μl used to transform competentHB101 cells. The resultant plasmid was called pSP31. Twenty μg of thisplasmid were digested with BamHI and a fragment of about 2900 bp wasisolated by gel electrophoresis, resuspended in TE and ligated to pCl/1previously cut with BamHI. This DNA (the pCl/1-pSP31-GAP-ADH2derivative) was used to transform HB101 and transformants with the BamHIcassette were obtained.

4.3.3.2. Transformation and Expression

Yeast strains 2150, PO17, and AB110 were transformed with thepCl/1-pSP31-GAP-ADH2 derivative, both short and long orientations. Thestrain 2150 gave no transformants. All other transformants were patchedon leu⁻ plates.

Yeast strain PO17 (Mat a, leu 2-04, cir°) was obtained by isolating aspontaneous revertant strain 21502-3 (Mat a, ade 1, leu 2-04, cir°). Toisolate the revertant PO17, 2150-2-3 yeast cells were grown in YEPD,washed in medium without adenine and about 6×10⁸ cells were plated ontosix adenine minus (ade⁻) plates. Four candidate revertants were testedfor other genetic markers by steaking on plates without uracyl (ura⁻),plates with no leucine (leu⁻), and minimal plates plus leucine. Growthwas observed on ura⁻ and minimal plus leu plates; no growth was observedon leu⁻ plates. Revertants were crossed with strain AB103.1 (Mat a, pep4-3, leu 2-3, leu 2-112, ura 3.52, his 4-580) to determine if thereversion was due to extragenic suppression. Based on tetrad analysis,none of the four independent ade⁺ revertants were due to extragenicsuppression. Based on good growth and high spore viability, one of therevertants was selected and named PO17.

Three different kinds of inductions were tried: (1). PO17 colonies wereinduced in either a 10 ml culture of YEP/1% glucose or a leu⁻/3% ethanolculture for 24 h. The yeast pellets were analyzed by both polyacrylamidegels and Westerns and even though the Coomassie-stained gel showed anegative result, the Western did light up a band of the correctmolecular weight with both induction methods. (2). PO17 colonies wereinduced in a 30 ml culture of YEP/1% ethanol for 48 h. Aliquots wereanalyzed by PAGE at various time points during the induction. TheCoomassie-stained gel shows a band in the correct molecular weight range(47-50 kd) that appears after 14 h in YEP/1% ethanol and reaches amaximum intensity at 24 h of induction. The Western result correlateswell with the Coomassie-stained gel, showing strong bands at 24 and 48h. (3). AB110 colonies were induced in either leu⁻/3% ethanol or YEP/1%glucose for 24 h. PAGE and Westerns were run and the results werenegative for the PAGE and positive for the Western, in both inductionmethods.

Expression and immunoreactivity were characterized as described below.

Cells from one patch PO17 (pCl/1-pSP31-GAP-ADH2) were inoculated in 50ml of leu⁻/7.1% glucose and grown overnight at 30° C. The saturatedculture was inoculated into 500 ml of the same leu⁻/7.1% glucose mediumand incubated overnight at 30° C. The saturated 500 ml culture was usedto inoculate a 10 L fermenter with YEPD. Cells were then harvested aboutfive days later.

A sample of cells from the fermenter were analyzed by PAGEelectrophoresis to determine expression levels of p31-SOD. In addition,Western analysis using a Trimar serum was performed on the samples todetermine its immunoreactivity.

The following procedure was used to prepare samples for PAGE andWesterns:

a. Cells (4 g) were resuspended in 7 ml of lysis buffer (50 mM Tris-HCl,pH 8.0, 1 mM EDTA, 150 mM NaCl, 1 mM PMSF) in a centrifuge tube and 4 mlof yeast-size glass beads were added.

b. Cells were vortexed at top speed in a VWR vortex for 10 minutes (1min. on ice, 1 min. vortexing).

c. The cell lysate was centrifuged at 18,000 rpm (39,000×g) for 10minutes in a JA 20 rotor. Both insoluble (pellet) and soluble(supernatant) fractions were further analyzed.

d. The supernatant was diluted in sample buffer (¼ dilution), boiled 10minutes and cooled to room temperature.

e. The pellet obtained in “c” was boiled during 10 minutes in samplebuffer (5 ml) and cooled to room temperature. The mixture wascentrifuged at 18,000 rmp (39,000×g) for 15 minutes in a JA 20 rotor.The supernatant was recovered and was diluted ¼ in sample buffer.

f. Samples from the supernatant (obtained in “d”) and solubilized pellet(obtained in “e”) were loaded on a 12% PAGE. A band corresponding toSOD-p31 (between MW 68 and 43 Kd) was present in the insoluble fraction(samples of solubilized pellet).

Immunoreactivity of SOD-p31 band was tested by Western Analysis usingserum. Trimar 0036 from an AIDS patient. For this purpose, proteins werefractionated on a 12% PAGE as previously described and transferred tonitrocellulose filter paper. The filter paper was then treated withserum 0036, followed by a second goat anti-human antibody conjugatedwith horseradish peroxidase (HRP). Color was developed using an HRPsubstrate. Bands corresponding to p31-SOD were present in samplescorresponding to the insoluble (Pellet) fractions. Bands of smallersize, present in both soluble and insoluble fractions, also react withthe sera and most probably correspond to degradation products ofSOD-p31.

4.3.3.3. Purification and Characterization

Frozen bacteria (Section 3.5) or yeast cells expressing the p31-SODfusion protein are thawed at room temperature and suspended in 1.5volumes of lysis buffer (20 mM Tris-Cl, pH 8.0, 2 mM EDTA, 1 mM PMSF,for bacteria; 50 mM Tris-Cl, pH 8.0, 2 mM EDTA, 1 mM PMSF for yeast),and mixed with 1 volume of acid-washed glass beads.

Cells are broken for 15 min in a non-continuous mode using the glasschamber of a Dynomill unit at 3,000 rpm, connected to a −20° C. coolingunit. Glass beads are decanted for 2-3 min on ice, the cell lysate isremoved. The decanted glass beads are washed twice with 30 ml of lysisbuffer at 4° C. The cell lysate is centrifuged at 39,000×g for 30 min.

The pellet obtained from the above centrifugation is washed once withlysis buffer, after vortexing and suspending it at 4° C. (samecentrifugation as above). The washed pellet is treated with 0.2% SDS(for bacteria) or 0.1% SDS (for yeast) in lysis buffer and agitated byrocking at 4° C. for 10 min. The lysate is centrifuged at 39,000×g for30 min. The pellet is boiled in sample buffer (67.5 mM Tris-Cl, pH 7.0,5% B-mercaptoethanol, 2.3% SDS) for 10 min and centrifuged for 10 min at39,000×g. The supernatant is recovered and further centrifuged at100,000×g for 60 min (60 Ti rotor). This step is replaced by a 0.45 μmfiltration when yeast is used. The supernatant from the abovecentrifugation is loaded (maximum 50 mg of protein) on a gel filtrationcolumn (2.5×90 cm, ACA 34 LKB) with a flow rate of 0.3-0.4 ml/min,equilibrated with phosphate-buffered saline (PBS), 0.1% SDS. Thefractions containing SOD-p31 are pooled and concentrated either byvacuum dialysis or using a YM5 Amicon membrane at 40 psi. The protein isstored at −20° C. as concentrated solution.

Gel electrophoresis analysis shows that the SOD-p31 protein migrateshaving a molecular weight of about 46 kd and is over 90% pure.

4.4. Reverse Transcriptase (RT)

The AIDS retroviral reverse transcriptase (RT) is an RNA-dependent DNApolymerase found in virions in low quantities. RT is encoded within adomain of the viral pol gene. The mature enzyme is derived byproteolytic processing from a large polypeptide precursor whose cleavageis thought to be mediated by a viral protease.

The amino terminal sequence of HIV RT has been reported [Veronese et al.(1986) Science 231:1289-1291] and corresponds to proline-156 of thepolymerase gene (nucleotide 2403, FIG. 5). The carboxy terminal end ofthe RT gene is estimated to be at valine-691 (nucleotide 3708).

4.4.1. pAB24/RT4 Expression Vector

A 6.1 kb EcoRI fragment of cloned proviral DNA was cloned into pUC19 atthe EcoRI site and designated pUC-ARV8A. Plasmid pUC-ARV8A was digestedwith BalI and KpnI which liberates a 1535 bp fragment containing codingsequence for amino acids proline-180 through tryptophane-690. Thisfragment was extended, using synthetic oligonucleotides, to includeproline-164 at the N-terminus and alanine-693 at the C-terminus. Thesynthetic DNA also provides a methionine initiation codon.

The RT-encoding fragment was modified by the addition of syntheticoligonucleotide adapters. The 5′ synthetic DNA has the followingsequence:

5′-CATGCCTATCTCTCCAATCGAAACCGTC     3′-GGATAGAGAGGTTAGCTTTGGCAG   CCAGTCAAGCTTAAACCAGGTATGGATGGG    GGTCAGTTCGAATTTGGTCCATACCTACCC   CCCAAGGTCAAGCAGTGG-3′    GGGTTCCAGTTCGTCACC-5′The 3′ adaptor was a KpnI/SalI adaptor with an in-frame stop codonhaving the following sequence:

    5′-CAGCATAG-3′ 3′-CATGGTCGTATCAGCT-5′The 5′ adaptor contains a HindIII site within the initiation codon sothat digestion with HindIII and SalI can facilitate subsequent cloningof the RT sequence into additional expression vectors.

The synthetic linkers and the cloned DNA were ligated by standardtechniques into the vector pPGAP1 (Section 4.2.1.1) which had beenpreviously digested with NcoI-SalI and gel isolated. The resultingexpression cassette containing the GAPDH promoter, the RT gene, andGAPDH terminator was excised with BamHI and cloned into pAB24 which hadbeen previously digested with BamHI and treated with alkalinephosphatase. The resulting expression plasmid was designated pAB24/RT4and is shown in FIG. 20.

4.4.2. Transformation and Expression

Plasmid pAB24-RT4 was used to transform yeast strain AB110, and leucineprototrophs grown in leucin-deficient media to 3 ml, followed by growthin YEPD to the 1 l level. Travis et al. (1985) J. Biol. Chem.260:4384-4389.

4.4.3. Purification

Cells from a 1 l culture were pelleted by centrifugation at 2,500 rpmfor 10 min. The cell pellet was resuspended in 300 ml of 50 mM Tris-HCl,pH 7.5, 14 mM B-mercaptoethanol, 1.2M sorbitol, and 200 μg/ml Zymolyase.Spheroplast formation, monitored by light microscopy, was allowed toproceed for 90 min at 30° C. After a low speed centrifugation, thepelleted spheroplasts were lysed in 40 ml of a buffer containing 50 mMTris-HCl, pH 7.5, 0.1% Triton X-100, and 1 mM DTT at room temperature.The yeast lysate was clarified by centrifugation at 20,000 rpm for 2hours and the supernatant was fractionated by stepwise NH₄SO₄precipitation. Greater than 90% of RT activity was in the 0-30% NH₄SO₄insoluble fraction. This NH₄SO₄ pellet was resuspended in 20 ml ofreverse transcriptase buffer (RTB; 50 mM Tris-HCl, pH 7.5, 2 mMB-mercaptoethanol, 0.2 mM EDTA, 0.1% Triton X-100, 20% vol/vol glycerol)containing 50 mM KCl. An Amicon pressure filtration device was used fordesalting. The extract was then applied to a cellulose phosphate column(Sigma C-2383) (2.5 cm×30 cm) preequilibrated in RTB containing 50 mMKCl. The column was washed with 100 ml of the same buffer. A lineargradient of 50 to 800 mM KCl in RTB was used for elution. Individualfractions were monitored for RT activity. The peak fractions of RTactivity were between 150 and 225 mM KCl. This material (about 15 mltotal) was pooled, desalted by Amicon filtration using RTB containing 50mM KCl, and then applied to a single-stranded DNA cellulose column(Sigma D-8273) (1.0 cm×10 cm) preequilibrated in RTB at 50 mM KCl. Thecolumn was washed with 30 ml of this buffer and eluted with a lineargradient of RTB from 50 to 800 mM KCl. Fractions were monitored for RTactivity and peak fractions were pooled.

4.4.4. Electrophoresis and Immunoblotting

Recombinant RT was analyzed by polyacrylamide gel electrophoresis andimmunoblotting techniques using AIDS patients' sera. The recombinantprotein gave an apparent gel mobility of approximately 66 kD, indicatingan extremely close approximation to the native p66 species, and in goodagreement with the calculated molecular weight of 62.5 kD. It was alsonoted that during purification, processing of this 66 KD proteinoccurred giving a second major species with an estimated molecularweight of 51±1.5 KD. This processing is presumably due to a yeastprotease since the region thought to encode ARV protease was notincluded in this expression construction. Previously, HIV gag-polfusions expressed in yeast were shown to be processed when this proteaseregion was included. Kramer et al. (1986) Science 231:1580-1584. Thatthe p66 and p51 species produced in yeast had identical N-termini(Pro.Ile,Ser.Pro.Ile, etc.) was confirmed by gas phase sequence analysisof the purified proteins (15 cycles; as a mixture, Panel A, lane 3).Thus, yeast may mimic the natural maturation processes for HIV RTs,giving rise to both p66 and p51. The sequence analysis also showed thatthe N-termini methionine derived from the synthetic initiation codon wasremoved in vivo. Interestingly, the processing observed in yeast mayindicate that HIV protease is not necessarily required for thisprocessing event in vivo. Preliminary immunoblot analysis of recombinantRT also indicates a high degree of reactivity with AIDS sera. Of 20 seratested, 19 scored positive.

4.4.5. RT Activity Assay

Analysis of recombinant RT activity by enzymatic assay was performed oncrude yeast lysates and the purified enzyme. Cruse lysate was preparedas follows. Cells from 25 ml culture were pelleted by centrifugation at2,500 rpm for 10 min. The cell pellet was resuspended in 7.5 ml of 50 mMTris-HCl, pH 7.5, 14 mM B-mercaptoethanol, 1.2M sorbitol, and 200 μg/mlZymolyase. Spheroplast formation, monitored by light microscopy, wasallowed to proceed for 90 min at 30° C. After a low speedcentrifugation, the pelleted spheroplasts were lysed in 1 ml of a buffercontaining 50 mM Tris-HCl, pH 7.5, 0.1% Triton X-100, and 1 mM DTT atroom temperature. The yeast lysate was clarified by centrifugation.

Using the assay conditions described for RT isolated from virions[Veronese (1986) Science 231:1289-1292], the relative activity ofyeast-derived RT was assayed using various primer template combinations.Thus, relative to (dT)˜₅·(rA)_(n) (100%), the enzyme activity with(dT)˜₅·(dA)_(n) was 4.3%, with (dG)˜₁₅·(rC)_(n) was 71.1%, and with(dG)˜₁₅·(rC^(m))_(n) was 2.4%. Yeast extracts form cells containingcontrol plasmids gave background levels of incorporation, excluding thepossibility of host-encoded RT activity. In all cases, the enzymereactions were linear for greater than 90 minutes. The results are shownin the following table:

Time cpm Incorporated 10′ 860 20′ 2110 30′ 2720 40′ 4046 50′ 3834 60′5360 90 8284 120′ 10626 160′ 11814 180′ 11750Kinetics of recombinant reverse transcriptase activity: 6 μl of RTsample incubated at 37° C. with 50 mM Tris-HCl, pH 7-8; 10 mM MgCl₂; 10mM DTT; 0.1M NaCl; 0.1 mM [³H] dTTP and 15 μg of (dT)˜₁₅·(rA)_(n) in atotal volume of 300 μl. 25 μl aliquots were removed at indicated timesand trichloroacetic acid precipitated on glass filters, washed, anddried, and their radioactivity was determined with a Beckmanscintillation counter.4.5. p25gag

4.5.1. Host-Vector System

Protein p25gag is synthesized by Saccharomyces cerevisiae AB110transformed with plasmid pCl/1-p25-ADH-GAP. Plasmid pCl/1-p25-ADH-GAP isa yeast expression vector which contains the sequence coding for p25gag[Sanchez-Pescador et al. (1985) Science 227:484] as well as pBR322sequences including the ampicillin-resistant (amp^(R)) gene and 2 micron(2μ) sequences [Broach, in Molecular Biology of the Yeast Saccharomyces,Vol. 1, p. 445 (Cold Spring Harbor Press, 1981)], including the yeastleucine (leu) 2-04 gene.

Expression of p25 is non-constitutive and it is under regulation of ahybrid ADH-2/GAPDH promoter derived from promoter sequences of the yeastalcohol dehydrogenase gene (ADH2) [Beier et al. (1982) Nature300:724-728] and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [EPOPub. No. 120,551] and of the GAPDH terminator. Induction of p25expression is achieved by low concentration of glucose in the growthmedium. Yeast strain S. cerevisiae AB110 has the following genotype: Matα, ura3-52, leu2-04, or both leu2-3 and leu2-112, pep4-3, his4-580,cir°. This strain was obtained as described below.

4.5.2. Saccharomyces cerevisiae AB110

Yeast strain S. cerevisiae 2150-2-3 (available from Lee Hartwell,University of Washington) was crossed with yeast S. cerevisiae strainAB103.1 transformant containing pCl/1 derivative. The diploids weresporulated and the tetrads dissected. Strains were maintained on leucineselective plates in order to ensure maintenance of the plasmid, sincethe parents are auxotrophs. A series of colonies were screened for theirgenotype with respect to a number of markers (Mat α, ura3, leu2,pep4-3).

The strain AB110 has the following genotype: Mat α, ura3-52, leu2-04 orboth leu2-3 and leu2-112, pep4-3, his4-580, cir°, and is obtained bycuring the above strain AB110 (pCl/1 derivative) of its resident plasmidby growth in the presence of leucine (absence of selective pressure) andthen selection for leu⁻ colonies by replica plating.

4.5.3. pCl/1-p25-ADH-GAP

Plasmid pCl/1-p25-ADH-GAP is a yeast expression vector which contains an“expression cassette” (see below) for p25gag cloned into the BamHI siteof vector pCl/1. Vector pCl/1 was previously described (Section 4.1.2).

An “expression cassette” for p25gag consists of the following sequencesfused together in this order (5′ to 3′): yeast hybrid ADH-2/GAPDHpromoter, a gene for p25gag, and GAPDH terminator. FIG. 21 shows aschematic of the construction of pCl/1-p25-ADH-GAP. The fragment shownat the top of the figure, containing p25gag, was constructed from ARV-2DNA, in which the sequences coding for leu (position 7) to an SphI siteat gly (position 90) (see FIG. 22 for amino acid numbers) were repeatedby synthetic DNA. A PstI site (encircled in the figure) was inserted atleu (position 7) by replacing the natural CTA with a CTG codon. Anatural PstI site at Ala79 was removed by making a silent third positionchange (dA to dC) in this codon. Two stop codons (indicated by an arrow)and a SalI site (circled) were placed adjacent to the C-terminal leu(position 232) codon by substituting synthetic DNA from the ScrFI siteat pro (position 225) to the SalI using synthetic oligonucleotides. Anadditional methionine codon (indicated with an asterisk) was used as theinitiation codon. The PstI-SalI fragment had been previously cloned inthe construction of pGAG25-10 (see Section 3.1.2. and FIGS. 7 & 8).

To construct the yeast expression vector, a 682 bp PstI-SalI fragmentcontaining the p25gag gene was isolated from plasmid pGAG25-10 (Section3.1.2) and ligated with a synthetic NcoI-PstI linker, which has thefollowing sequence:

  Met Pro Ile Val Gln Asn Leu Gln 5′                               3′ CATG CCT ATA GTG CAG AAT CTG CA    169/24 (Nco) GGA TAT CAC TGC TTAG  Pst    170/16       3′                  5′This was cloned into pBS100 (Section 4.3.2) which had been previouslydigested with NcoI and SalI and gel isolated. The BamHI expressioncassette was excised from the resulting plasmid and ligated into BamHIdigested and phosphatased treated pCl/1 to yield pCl/1-p25-ADH-GAP. Thep25gag produced in yeast differs from that produced in E. coli (Section3.1.) by the presence of the naturally occurring proline after themethionine at the N-terminus.

FIG. 22 shows the nucleotide sequence of the p25gag insert cloned inpCl/1-p25-ADH-GAP and the amino acid sequence derived from it. DNAsequences that are not underlined in the p25 region are derived directlyfrom the ARV-2 proviral DNA. Underlined sequences were chemicallysynthesized by the phosphoramidite method as originally described byBeaucage & Caruthers, (1981) Tetrahedron Lett. 22:1859.

4.5.4. Transformation and Expression

Yeast cells were transformed following the procedure of Hinnen et al.(1978) Proc. Natl. Acad. Sci. USA 75:1929. The transformation mix wasplated onto selective leu⁻ agar plates. Plates were incubated at 30° C.for 2 to 4 days.

Single transformant colonies were transferred into leu⁻, 8% glucosemedium and grown at 30° C. until saturation. For induction ofexpression, a 1/25 dilution of the saturated culture into YEP/1% glucosewas made and the cells were grown to saturation. Cells were harvested,lysed with glass beads and the insoluble material was collected bycentrifugation. The pellet was resuspended in gel sample buffer andboiled. Extracts were fractionated on standard denaturing acrylamidegels. Laemmli (1970) Nature 227:680. Proteins were visualized bystaining with Coomassie blue.

The extent of expression was initially determined by appearance of a newprotein of the expected molecular weight in extract of transformantsharboring pCl/1-p25-ADH-GAP as compared with control extracts (cellstransformed with vector without p25gag insert). Immunoreactivity of thep25 protein was determined by standard western analysis [Towbin et al.(1979) Proc. Natl. Acad. Sci. USA 76:4350] using human serum from anAIDS patient. The western analysis showed immune reaction of a proteinof about 25,000 daltons. No reaction was observed with non-immune serum.

For preparation of a master seed stock, transformant cells were streakedonto a leu⁻ plate and incubated at 30° C. for 2 days. Five singlecolonies were picked and individually inoculated into 5 ml of leu⁻, 8%glucose liquid medium and grown overnight at 30° C. A 1 ml aliquot wasused to test expression of p25 and 15% glycerol was added to the rest ofeach culture. Glycerol cell stocks were aliquoted into 1 ml vials,labeled and quickly frozen in liquid nitrogen. Aliquots of the culturecorresponding to the highest expressor were selected as the master seedstock and stored at −70° C. The seed stock was tested for absence ofbacterial contamination.

4.5.5. Protein Purification.

Cells from the patch which gives highest expression of p25 were used toinoculate 40-60 ml of leu⁻, 7.1% glucose medium. Cultures were grown tosaturation at 25-35° C. with agitation. An aliquot (10-50 ml) of thefirst culture were added to 400-600 ml of leu⁻ 7.1% glucose medium.Cultures were grown to saturation at 25-35° C. with agitation. Analiquot (100-500 ml) of the second inoculum was added to 8-12 L ofYEP/1% glucose containing 1 ml of antifoam in a 16 L fermentor. Cultureswere grown for 24 to 48 hours at 25-35° C. with agitation. Followingfermentation, the approximately 8-12 L of yeast culture were centrifugedthrough a continuous flow centrifuge, and the cells were harvested.

Frozen (or refrigerated) packed cells (50-150 g portions) were thawedand suspended in Lysis Buffer (1 mM PMSF, 2 mM EDTA, 150 mM NaCl, 50 mMTris-HCl, pH 8.0; total volume of cells and Lysis Buffer=280 ml), and160-170 g of acid-washed glass beads were added. Cells were broken in anon-continuous system using a 300 ml glass unit of a Dyno-mill at3,000±500 rpm for 10-25 minutes. The exterior jacket temperature of theDyna-mill was maintained at −15° to −20° C. by an ethylene glycolsolution. Glass beads were allowed to settle by letting the mixture siton ice. The cell lysate was decanted. Glass beads were washed with LysisBuffer and the wash was added to the cell lysate. The lysate wascentrifuged at: 39,000×g (30-70 minutes, depending on rotor) for 30minutes. The pellet was discarded, and the soluble fraction was furthercentrifuged at 100,000×g for 60 minutes at 2-8° C. The fatty layer(about ¼ of total volume) was aspirated off. The supernatant wasdecanted, and the pellet was discarded.

The soluble fraction obtained in the previous step was diluted ten foldby adding 9 volumes of 0.03 M Tris-HCl, 1 mM EDTA, pH 9.0. The pH of thediluted soluble fraction was adjusted to 9.0±0.5, then the material waschromatographed at 4-8° C. on a DEAE Sephacel column equilibrated with0.03 M Tris-HCl, 1 mM EDTA, pH 9.0. Material was eluted using the samebuffer; absorbance of the eluate was monitored at 280 nm, and 20-25 mlfractions were collected. Fractions were assayed by SDS-PAGE, and thosecontaining p25 protein were pooled.

The pooled fractions were concentrated to a protein concentration of20-25 mg/ml by ultrafiltration in an Amicon unit under N2 pressure. Theconcentrate was chromatographed on an ACA54 (LKB) column equilibratedwith at least 1 column volume of 0.03 M Tris HCl, 1 mM EDTA, pH 9.0.Material was eluted using the same buffer; absorbance of the eluate wasmonitored at 280 nm, and 10-30 ml fractions were collected. Fractionscontaining p25 protein as determined by SD-PAGE were pooled. Thefraction pool was concentrated to approximately 1-2 mg/ml total proteinby ultrafiltration in an Amicon unit under N2 pressure. Proteinconcentration of the Bulk p25 was adjusted to 1.2-1.8 mg/ml (based onLowry assay).

4.6. p53gag

The gag proteins from the HIV retrovirus are derived from a gagprecursor polypeptide, designated p53gag. By combining HIV DNA derivedfrom pUC-8A, (a subclone containing the insert from λARV-2 (8A) clonedinto pUC19) with synthetic oligonucleotides and cloning into a yeastexpression vector the complete p53gag precursor protein was synthesizedby yeast.

4.6.1. Construction of pCl/1-GAP-p53 A 1424 bp SacI-BglII fragment wasgel isolated from pUC-8A. This fragment corresponds to nucleotides 225to 1650 as shown in FIG. 5. The SacI-BglII fragment was further digestedwith HgaI and then ligated with the following oligonucleotide linker:

5′-GCCCTTTGGGAAACCAT-3′

3′-CGGGAAACCCTTTGGTACCCAC-5′

The ligated linker fragment was digested with NcoI and BglII and thencloned into pPGAP-IGF1 which had previously been digested with NcoI andBglII. The NcoI site of the linker functions to regenerate theN-terminal methionine of p53. Plasmid pPGAP-IGF1 is plasmid pPGAP1 asdescribed in Section 4.2.1.1. with a 220 bp NcoI-SalI fragment encodingIGF-I. The IGF-I sequences only serve as a matter of convenience due tothe presence of a BglII site 60 bp from the 3′ end of the insert. Theresulting plasmid, pPGAP-p53/IGF1, was digested with BglII and SalI andligated with a 201 bp synthetic oligonucleotide fragment which encodesthe C-terminus of the p53 protein to give plasmid pPGAP-p53. The 1505 bpNcoI-SalI fragment contains the entire coding region of p53 from aminoacid methionine-1 to glutamine-502. Plasmid pPGAP-p53 was digested withBamHI to isolate the expression cassette containing the GAPDHpromoter-p53 gene-GAPDH terminator as a 2843 bp BamHI fragment which wasthen cloned into pCl/1 which had previously been digested with BamHI.The resulting plasmid was called pCl/1-GAP-p53.

4.6.2. Transformation and Expression

S. cerevisiae strain JSC302 was constructed by transforming strain AB116to G418 resistance with plasmid pDM15 (FIG. 14). Plasmid pDM15 consistsof Kluyveromyces lactis ADH1 promoter and terminator sequences flankingthe G418 gene, pBR322 sequences, and a GAP promoter-ADR1 expressioncassette. This integrating plasmid was targeted to the ADR1 locus.

Plasmid pCl/1-GAP-p53 was transformed into yeast strain S. cerevisiaeJSC302 following the procedure of Hinnen et al. (1978) Proc. Natl. Acad.Sci. USA 75:1929. The transformation mix was plated onto selective leu⁻agar plates. The plates were incubated at 30° C. for 2 to 4 days. Singletransformant colonies were transferred into leu⁻, 8% glucose medium andgrown at 80° C. until saturation. For induction of expression, a 1/25dilution of the saturated culture into YEP, 1% glucose was made and thecells-were grown to saturation. Cells were harvested, lysed with glassbeads and the insoluble material was collected by centrifugation. Thepellet was resuspended in gel sample buffer and boiled. Extracts werefractionated on standard denaturing polyacrylamide gels. Proteins werevisualized by staining with Coomassie blue dye. A band of theappropriate molecular weight for p53 was present in the culturestransformed with pCl/1-GAP-p53 but not in control extracts of JSC302.

4.6.3. Protein Purification

Cultures of JSC302 (pCl/1-GAP-p53) were grown under the conditionsdescribed above except that the culture in YEP, 1% glucose was allowedto grow for only 24 hours. Cells were harvested and processed using theglass bead lysis procedure with Triton lysis buffer. Triton Lysis Buffer(0.1%, Triton X-100 (10 mM Tris HCl 8.0), 62.5 mM EDTA pH 8.0, 50 mM,Tris HCl pM 8.0). The proteins were precipitated with 40% NH₂SO₄. Thepellet was resuspended in H_(x)O and dialyzed with 50 mM phosphate pH7.0, 1 mM EDTA, 1 μg/ml Leupeptin, 1 μg/ml Leupeptin, 1 μg/ml Aprotinin,1 mM PMSF (Phenylmethyl-sulfonylfluoride).

The solution is applied to a Mono-Q resin (Pharmacia) on a FPLC column(Pharmacia). The column is eluted with a 0-1M NaCl gradient. Fractionsare collected and analyzed by Coomassie staining western blot analysisfor the presence of p53. The peak fraction is made up to 20% glycerol.The column may be repeated to obtain higher purity of the protein.

5. Immunoassay for Anti-HIV Abs Using Recombinant HIV Polypeptides

Diagnostic assays based on the ELISA technique and employingrecombinantly produced viral antigens have been developed for thedetection of antibodies to HIV. Micro titer plate based ELISAs and animmunoblot strip ELISAs have been configured which use either three orfour recombinant viral antigens: the major core protein p25gag, theendonuclease region of the viral polymerase gene p31pol, and one or twopolypeptides from the envelope gene, either from gp120 and/or gp41coding regions.

5.1. ELISA-A

5.1.1. Assay Protocol

Stock solutions of purified p25gag protein (Section 3.1.5) (1.25 mg/mlin 20 mM sodium phosphate, 0.1% SDS, pH 7.2), purified env-2 protein(Section 4.2.2) (2 mg/ml in 20 mM sodium phosphate, 0.1% SDS, pH 7.2),and purified SOD-p31 fusion protein (Section 4.3.3) (2 mg/ml in 20 mMsodium phosphate, 0.1% SDS, pH 7.2) were prepared.

For coating microtiter plates (Dynatech Immulon I), 1 part each of thestock solutions of p25gag, env, and SOD-p31 were added to 997 parts ofborate coating buffer (0.05 M borate, pH 9.0). One hundred microlitersof the coating solution was added to each well, and the plates werecovered and incubated 2 h at 37° C. or 12 h at 4° C. The coatingsolution was then aspirated from the wells and the plates washed 6× withwash solution (0.137 M 0.8% NaCl, 0.05% Triton X-100).

Serum samples were diluted 1:100 in dilution solution (0.1% casein, 1mM. EDTA, 1% Triton X-100, 0.5 M NaCl, 0.01% thimerosal, pH 7.5) withyeast protein (strain AB103.1) extract (1:40 dilution, approximately 2mg protein per ml in PBS containing 1% Triton X-100, 2 mM PMSF, 0.01%thimerosal) and E. coli protein extract (1:40 dilution, approximately 1mg protein per ml in PBS containing 1% Triton X-100, 2 mM PMSF, 0.01%thimerosal) added to the dilution solution. Extraction procedures weresimilar to those described in 16 and 18 above but using nonrecombinantstrains. One hundred microliters of diluted serum was added to each welland incubated 30 min at 37° C. The plates were then washed 6× with washsolution.

Goat anti-human Ig labeled with horseradish peroxidase (Cappel) diluted1:8000 in dilution solution without added yeast and E. coli extractswere added at 100 μl/well to the plates and incubated 30 min at 37° C.The plates were then washed 6× with wash solution. Substrate solution(10 ml citrate buffer, 10.5 g citric acid/liter dH₂O, pH to 4.0 with 6 MNaOH), 0.1 ml ABTS [15 mg/ml 2,2′-azino-di-(3-ethylbenzthiazolenesulfonic acid) in dH₂O] and 3.33 μl H₂O₂ at 100 μl/well was then addedto the plates and the plates wrapped in foil and incubated at 37° C. for30 min. The reaction was then stopped by adding 50 μl/well of 10% SDS.Readings were made with a Dynatech ELISA reader set for dual wavelengthreading: absorbance wavelength of 1 (410 nm) and reference wavelength of4.

5.1.2. Results

The following sera were tested:

A. 89 consecutive blood donors from the Kansas City Blood Bank (“normalblood donors”): log nos. 1001-1081, 1085-1092.

B. 52 sera from patients with lymphadenopathy syndrome (LAD) or AIDS orsexual partners of persons with LAD or AIDS (referred to as“contacts”)—all obtained from UCSF AIDS Serum Bank panel: log nos.4601-4652.

The positive/negative cut-off used was 5×(average background signalsignal with diluent alone) and was determined to be 0.195. Thus, serawith signals below 0.195 were rated (−); those above were rated (+).Each sample was also evaluated by the commercially available ABBOTT HTLVIII EIA kit (Abbott Labs) and by Western analysis.

Tests on the normal blood donor samples indicated all except one werenegative in the invention ELISA. This normal serum scored negative inthe ABBOTT HTLV-III EIA test, but was actually positive, as confirmed byWestern analysis.

The results of the tests on the 52 sera from LAD and AIDS patients andcontacts are tabulated below:

Serum ABBOTT Invention No. Diagnosis EIA ELISA Western 4601 Contacts +1.89 + + 02 Contacts − 0.04 − − 03 Contacts + 1.44 + + 04 Contacts +1.92 + + 05 Contacts − 0.04 − − 06 Contacts + >2 + + 07 Contacts +1.37 + + 08 Contacts + 1.60 + + 09 Contacts + >2 + + 10Contacts + >2 + + 11 Contacts + 1.94 + + 12 Contacts + >2 + + 13Contacts + >2 + + 14 Contacts + >2 + + 15 Contacts + 1.97 + + 16 AIDS +0.61 + + 17 AIDS + >2 + + 18 AIDS + >2 + + 19 AIDS + 1.58 + + 20 AIDS +1.58 + + 21 AIDS + 0.76 + + 22 AIDS + 1.74 + + 23 LAD + 1.26 + + 24LAD + >2 + + 25 AIDS + 1.04 + + 26 AIDS + 1.24 + + 27 AIDS + 1.40 + + 28AIDS − 0.07 − − 29 LAD + 1.93 + + 30 Contacts + 1.96 + + 31 AIDS +1.76 + + 32 AIDS + 0.90 + + 33 AIDS + 1.69 + + 34 LAD + 1.09 + + 35AIDS + 1.54 + + 36 AIDS + 1.22 + + 37 AIDS + 1.96 + + 38 AIDS − >2 + +39 LAD + 1.85 + + 40 LAD + >2 + + 41 LAD + 0.84 + + 42 LAD + 1.59 + + 43LAD + 1.71 + + 44 AIDS + 1.40 + + 45 LAD + >2 + + 46 AIDS + 1.38 + + 47AIDS + 1.29 + + 48 LAD +/− 1.93 + + 49 LAD +/− 0.48 + + 50 LAD − 0.04 −− 51 LAD − 0.07 − − 52 LAD + 1.92 + +

The above results show that the ELISA-A, using recombinant HIV proteins,is at least as good as the ABBOTT HTLV III EIA test or Western analysis.

In the ELISA-A reported in this example, the yeast and bacterialextracts were added to the serum to bind serum antibodies to yeast andbacteria to prevent such antibodies from binding to minor contaminantsin the recombinant HIV-1 protein preparations. Both yeast and bacterialextracts were required since the recombinant polypeptides includedpolypeptides expressed in yeast and polypeptides expressed in bacteria.If all the polypeptides were expressed in the same type of organism,only one extract would be needed. For instance, if a p25gag polypeptideexpressed in yeast was substituted for the bacterially produced p25gagpolypeptide of the example, only yeast extract would be added to theserum samples.

5.2. ELISA-B

5.2.1. Assay Protocol

Frozen stocks of the following purified proteins were thawed and used tomake a solution containing: p25gag (Section 4.5) 1.25 μg/ml, SOD-p31(Section 4.3.3) 1.00 μg/ml, SOD-env5b (Section 3.6) 1.25 μg/ml, andenv-2 (Section 4.2.2) 0.50 μg/ml in 0.05 M sodium borate, pH 9.0. Forcoating microtiter plates, 100 μl of the above solution was pipettedinto each well of Immulon I round bottom microtiter plates (DynatechLaboratories), and incubated for 2 h at 37° C. The coating solution wasthen aspirated from the wells. The plates were washed three times with200 μl/well Wash Buffer [100 mM Sodium Phosphate pH 7.4, 140 mM SodiumChloride, 0.1% Casein (Sigma), 0.05% Triton X-100 (Sigma), 0.01% (w/v)Thimerosal (Sigma)] and then washed two times with 200 μl/well PBS (10mM Sodium Phosphate pH 6.7, 150 mM Sodium Chloride). The plates werethen post coated by incubating with 200 μl/well of Postcoat solution[PBS, 0.1% (w/v) Casein, 2 mM PMSF (phenylmethyl-sulfonylfluoride)] for30 minutes at 15-30° C. The Postcoat solution was aspirated off. Theplates were dried in a lyophilizer (such as: Virtis Unitop 600 SLlyophilizer, Virtis Company) overnight or by incubating for 2 h at 25°C.

To assay samples in the ELISA-B assay, 100 μl of Samples Diluent [100 mMSodium Phosphate, pH 7.4, 0.5 M Sodium Chloride, 1 mM EDTA, 0.1% casein,1% Triton X-100, 100 μg/ml yeast extract (see below), 100 μg/ml E. coliextract (see below), and 0.01% Thimerosal] was added to each of thecoated wells on the plates. Ten μl of the samples to be assayed orcontrols were pipetted into the wells. The plates were then sealed andincubated for 1 h at 37° C. The sample was then aspirated and the plateswere washed three times with 200 μl/well of distilled water. The plateswere then incubated with a goat anti-human IgG-HRP (horseradishperoxidase) conjugated antibody (available from commercial sources,i.e., Tago or Cappel, or the conjugate may be synthesized by the Nakaneprocedure). The anti-human IgG-HRP conjugate was diluted 1/3200 inConjugate Solution I [PBS, 5% noral goat serum, 0.01% ANS(8-Anilino-1-naphthalene sulfonic acid, ammonium salt), 0.01%Thimerosal]. A final dilution to 1/32000 was made in Conjugate DiluentMT [116 mM Sodium Phosphate, pH 7.4, 0.622 M Sodium Chloride, 0.56% BSA,2.22% normal goat serum, 1.11% Triton X-100, 0.11% Casein, 0.0044% ANS,0.01% Thimerosal] immediately before use. One hundred μl of the dilutedconjugate was added per well (except for blank wells). The plates weresealed and incubated for 1 h at 37° C. The conjugate solution wasaspirated off and the plates were washed three times with 200 μl/well ofdistilled water. Then 100 μl of Developer [made fresh: 50 mM SodiumCitrate adjusted to pH 5.1 with 1 M Phosphoric Acid, 0.6 μl/ml 30% H₂O₂,OPD tablet (Sigma) (1 tablet/5 ml of buffer)] was added to each well andincubated thirty minutes at room temperature (15°-30° C.). The reactionwas stopped by the addition of 50 μl of 4N H₂SO₄ to each well.

The microtiter plates were analyzed on a standard ELISA reader (such asBiotek EIA Autoreader Model EL310, Biotek Instruments) by reading theabsorbance of developed color at 492 nm. The results were analyzed bycomparing the values generated for the samples against an assay cutoffvalve (cutoff=0.5×average absorbance value for the positive controls.)Yeast and E. coli extracts used in the Sample Diluent were prepared inan analogous manner to the purification process for recombinantpolypeptides in Sections 3.1.4.2-3.2.5 and 4.1.4.1-4.1.4.3, exceptnon-recombinant strains S. cerevisiae AB103.1 and E. coli D1210 wereused.

5.2.2. Results

The ability of ELISA-B to detect the presence of antibodies directedagainst HIV was compared to a licensed and commercially available ELISAproduced by DuPont.

493 serum samples were run in ELISA-B and the DuPont screening ELISA.The panel of sera was composed of specimens from various sources: 205samples from CDC (clinically categorized as AIDS, ARC, contact, falsepositive from licensed screening ELISA, and 4 negative); 101 samplesfrom UCSF (clinically categorized as AIDS, ARC, and contact); 187samples obtained from Interstate Blood Bank, Pa., (initially scoredpositive on licensed screening ELISA). In this group of samples therewere eight (8) discrepancies. Four (4) samples were found positive byELISA-B and negative in the DuPont ELISA. Correlation between the DuPontassay and ELISA-B was 98.4%. Concensus data indicated that of the four(4) samples found positive by ELISA-B and negative by the DuPont ELISA,three (3) (LW 47, 4202, and 4225) were true positives (DuPont ELISAfalse negatives) and one (4279) was an ELISA-B false positive. Concensusdata also indicated that the four samples found negative in ELISA-B andpositive in the DuPont ELISA (LW 12, 20061, 20145, 20162) were negative(DuPont ELISA false positives). For the eight (8) discrepant samplesELISA-B differed from the concensus data in one (1) case and the DuPontELISA differed in seven (7) cases. Also note that one sample was falsepositive in both assays.

The positivity or negativity of a specimen in these panels wasdetermined from the concensus results of commercial viral ELISAs, themicrotiter plate assay using recombinant antigens, the strip ELISA,Western Blot data (when available) and clinical data, if available.

TABLE Correlation of ELISA-B with the DuPont Screening ELISA DiscrepantSamples

S/CO = signal: cut off ratio if = or > 1.0 samples is positive5.3. Dot Blot Assay

Nitrocellular strips (0.5×5 cm) are spotted with 50 ng polypeptide inPBS (spotting volume 2 μl). After spotting the strips are dried at roomtemperature for 1 h or more. The strips are then post-coated in a 5%solution of Carnation non-fat dry milk in PBS, 0.01% Thimerosal, for15-60 min at room temperature. Each test solution sample is diluted 1:50in 0.5 ml of the post-coating solution in a test tube. A post-coatedstrip is then placed in the tube and incubated in the sample withrocking at 37° C. for 1 h. The strip is then removed from the tube andwashed with post-coating solution. The strip is then incubated for 15min at room temperature in goat anti-human Ig reagent labeled with horseradish peroxidase diluted 1:500 in post-coating solution. Afterincubation in the labeled antibody, the strip is washed serially withPBS, 1% Triton, and distilled water. The strips are developed byincubating them in substrate solution (see 23 above) for 15 min at roomtemperature.

Positive samples will cause a visually perceptible color change at thespotting site. Normal (negative) sera sample yield no color change orgive a faint signal that is discernible from a positive signal.Competition assays may be run on sera giving faint signals to verifythat they are negative. In the competition assay, polypeptide (10-25μg/ml) is added to the test sample and incubated from 1 h at 37° C.before the strip is incubated in the sample. With authentic positivesera the signal is completely blocked by the added polypeptide, whereaswith normal (negative) sera there is no change in signal.

5.4. Immunoblot Strip ELISA Assay

In the immunoblot strip ELISA, recombinant derived viral antigens areindividually coated in bands on a nitrocellulose strip and reacted withsamples to bind anti-HIV specific antibodies.

Frozen aliquots of the individual recombinant polypeptides were thawedand diluted to the appropriate concentration in Coating Solution [PBS pHadjusted to 7.4, 375 mg/l Napthiol Blue Black (used only as an inertmarker)] as follows: p25gag (Section 4.5) 3-4 μg/ml, SOD-p31 (Section4.3.3) 1-1.6 μg/ml, SOD/env-5b (Section 3.6) 1.0 μg/ml, env-2 (Section4.2.2) 0.5 μg/ml. The individual protein solutions were coated asindividual bands on a sheet of nitrocellulose paper; BA85 NC™;Schleicher & Schuell, Inc., Keene, N.H., (pre-wetted with PBS buffer)The coating apparatus can be any apparatus which allows for solutions tobe applied to filter paper under vacuum pressure as discrete bands, suchas Minifold® II Slot-Blot System; Schleicher & Schuell.

The coated sheets are dried overnight. The dried, coated sheets areimmersed for five minutes in Blocking Buffer (PBS, 1% (w/v) Casein,0.01% Thimerosal, pH adjusted to 7.4) and then dried overnight. Thecoated and blocked nitrocellulose sheets were attached to a backingsheet of paper with double faced tape and then cut into individualstrips which contain the individual bands corresponding to each of therecombinant viral antigens.

The sample to be assayed was diluted 1/100 with Sample Diluent by adding10 μl of the sample to 1 ml of Sample Diluent [PBS, 0.1% Casein, 1 mMEDTA, 2.0% Triton X-100, 1 mg/ml Yeast extract, 500 μg/ml E. coliextract, 0.2 μg/ml YP45 extract, 0.01% Thimerosal, pH adjusted to 7.4].The strips were then individually soaked in the diluted sample solutionwith agitation for 2 h at room temperature (15° to 30° C.). The stripswere removed from the diluted sample solution and washed four times withwater. The strips were then incubated with 1 ml/strip of Goat anti-humanIgG-HRP conjugate diluted in Conjugate Buffer [PBS pH 7.2, 0.3% Casein,5% normal goat serum, 0.01% ANS, 0.01% Thimerosal] with agitation for 30minutes at room temperature (15°-30° C.).

The strips were removed from the conjugate solution and then washedthree times with water. The strips were then incubated with 1. ml/stripof Developer Buffer [10 mM Sodium Phosphate, 20 mM Sodium Chloride, 0.8μl/ml 30% hydrogen peroxide, 0.05% (w/v) 4-chloro-1-naphthol, 16.6%methanol] with agitation for 15 minutes at room temperature (15°-30°C.). The reaction was stopped by removing the strips from the DeveloperBuffer and washing two times in water. The color developed on the bandis compared with positive control bands (IgG applied at 0.25 μg/ml and1.25 μg/ml).

Yeast and bacterial proteins were used to preabsorb antisera forcross-reactive antibodies. Yeast extract was prepared by processing bulkyeast S. cerevisiae (Red Star Yeast, grade 1; Red Star, Oakland, Calif.)through the standard lysis procedure as described previously for thepurification of yeast recombinant proteins. The insoluble cellulardebris was separated from the soluble protein fraction. The E. coliextract was prepared by growing E. coli strain D1210 (pSODCF2), lysingthe cells, and solubilizing the pellet as described in Section 3.6.4.YP45 extract is a yeast protein and was prepared by growing S.cerevisiae AB110 and following the purification procedure described inSection 4.3.3.3. A 45 kd yeast protein was purified which helps inpreabsorbing nonspecific cross-reactive antibodies found in some area.

6. Serology Studies with Recombinant HIV Polypeptides

6.1 Immunoblot with Env Polypeptides

Recombinant polypeptides originating from different regions of the envgene of the ARV-2 isolate of HIV (Section 4.2) were used to characterizethe anti-envelope antibody response of virus seropositive individuals.The sera characterized included specimens from AIDS patients, ARCpatients and clinically healthy homosexual men with documented exposureto the virus through sexual contact with AIDS or ARC patients(contacts).

Regions of the envelope gene of the ARV-2 isolate of HIV were clonedinto yeast expression vectors (env-1, env-2, env-3) or bacterialexpression vectors (env-5b), and used to produce recombinant proteins asdescribed in Sections 3 and 4.

Extracts of yeast for SDS polyacrylamide gel electrophoresis wereprepared as follows: 1 ml of yeast grown to 20 O.D. (A450) were pelletedand suspended in 200 μl of electrophoresis sample buffer. The mixturewas boiled for 10 min and then centrifuged at 12,000 g for 2 min toremove cell debris prior to electrophoresis. To prepare bacterialextracts for gel electrophoresis, bacterial cells from 1 ml of culturewere pelleted, suspended in 200 μl of electrophoresis sample buffer, anddisrupted by 3 cycles of freezing and thawing. The mixture was boiledfor 10 minutes prior to electrophoresis.

Yeast or bacterial extracts were electrophoresed on standard Laemmlidiscontinuous SDS-polyacrylamide (12% acrylamide) gels using a BioradLaboratories minigel apparatus. Laemmli (1970) Nature 227:680-685. Theextracts (100 μl) were loaded into an 8 cm well in the stacking gelprior to electrophoresis. Following electrophoresis, proteins weretransferred to nitrocellulose filters. Towbin et al. (1979) Proc. Natl.Acad. Sci. USA 76:4350-4355. Strips of the filter with theelectroblotted lysates were cut and each strip reacted with anindividual serum sample diluted 1/100.

Sera from 85 seropositive individuals, including AIDS patients, ARCpatients, contacts and clinically healthy individuals, as well as a poolof sera from normal human blood donors, were assayed on env-1, env-2 andenv-3. The blots were processed with Carnation nonfat dry milk asdescribed previously. Johnson et al. (1984) Gene Anal. Techn. 1:3-8. Theenzyme conjugate (diluted 1/200) was goat antiserum to humanimmunoglobulin G (Cappel Laboratories) and the chromogen was HRP colorreagent (4-chloro-1-Napthol; Biorad Laboratories).

Positive immunoblot assays obtained with env-1, -2, and -3 are shown inFIG. 30. A prominent band at 28,000 was seen in the env-1 blot with apatient's serum with anti-env-1 antibodies. In the immunoblot of env-2,the antibody-positive serum reacted with a protein of approximately 55kD. With env-3, the positive serum reacted with a series of bandsmigrating between 30,000 and 35,000. The multiple immunoreactive speciesin the env-3 immunoblot are due to differences in glycosylation (seeSection 4.2.3) of this polypeptide during its synthesis in yeast. Fivepotential glycosylation sites are contained within the coding region forenv-3.

The results of assays of the reactivity of sera obtained from thevarious patient groups in the 85 serum panel with the env-1, env-2 andenv-3 are presented in table below:

Clinical Number Number Reacting With: Group Tested env-1 env-2 env-3Contacts 21 4 (19%) 21 (100%) 21 (100%) ARC 26 3 (12%) 26 (100%) 26(100%) AIDS 38 6 (16%) 36 (95%) 36 (95%)

All of the specimens selected for this analysis were positive in anenzyme-linked immunosorbent assay (ELISA) for antibodies to the AIDSretrovirus. Weiss et al. (1985) J.A.M.A. 253:221-225. Sera from all ofthe contacts and ARC patients had antibodies that reacted with env-2 andenv-3 indicating that they had antibodies to both gp120 and gp41 thatcould be detected with these non-glycosylated polypeptides in immunoblotassays. Thirty-six of the 38 AIDS patients (95%) had antibodies thatreacted with env-2. The same thirty-six sera also reacted with env-3.The two AIDS sera that did not react in immunoblot assays with env-2 orenv-3 either lacked antibodies detected using these recombinantantigens, or their antibody titers were below the limit of detection inthese assays, at least in generating an immune response to sequentialepitopes.

When these sera were tested in immunoblot assays with env-1, only aminority in each diagnostic group scored positive. The percentage ofcontacts, ARC patients and AIDS patients with antibodies to env-1 was19%, 12% and 16%, respectively. Since env-1 corresponds to theamino-terminal half of env-2 (amino acids 26-491, the majority ofgp120), this observation suggests that the carboxyl terminal portion ofgp120 is much more immunogenic in infected individuals than theamino-terminal half of the polypeptide, at least in generating an immuneresponse to sequential epitopes.

Because the env-1 polypeptide was also represented in env-2, it couldnot be determined directly from the immunoblot results if env-1 positivesera reacted exclusively with env-1 or were also reactive with thecarboxyl-terminal half of env-2. To evaluate the reactivity of env-1positive sera with the carboxyl-terminal half of env-2, competitionexperiments were carried out. Sera were reacted with env-1 and env-2 inimmunoblot assays in the presence or absence of an excess of env-1 inthe serum diluent. An env-1 positive serum, diluted 1/100, was incubatedwith: env-1 immunoblot with no addition, env-1 immunoblot with 50 μg/mlenv-1 in diluent, env-2 immunoblot with no addition, env-2 immunoblotwith 50 μg/ml of env-1 in diluent and env-2 immunoblot with 50 μg/ml ofenv-2 in diluent. The reactivity of the env-1 serum sample with theenv-1 blot was completely eliminated by preincubation with env-1.However, reactivity with the env-2 blot was still evident. Similarresults were observed with all of the env-1 positive sera, indicatingthat they all reacted with the carboxyl terminal as well as the aminoterminal half of the gp120 polypeptide.

Immunoblots of extracts from bacteria expressing a subregion of gp41 (aa557-667) as an hSOD fusion, referred to as env-5b, with an AIDSpatient's serum, showed a prominent immunoreactive species at 32,000daltons that was absent from immunoblots of extracts from bacteriatransformed with the vector lacking the env insert (Section 3.6). Thiswas approximately the molecular weight expected for a polypeptide codedfor by the sum of the hSOD and env gp41 sequences. Twenty serum samplesshown previously to contain antibodies that reacted with env-3 inimmunoblot assays were reacted with env-5b immunoblots. All 20 specimensreacted with the fusion protein.

Immunoblot assays detect antibodies which recognize principallysequential determinants. The results presented here indicate that virusseropositive individuals frequently have mounted immune responses tosequential epitopes within the carboxyl terminal half of the gp120polypeptide. In addition, such individuals have also mounted immuneresponses to sequential epitopes within gp41 (env-5b). The prevalence ofantibodies to these two env regions detected by using recombinantpolypeptides from a single virus isolate suggests that there areepitopes within both regions that are highly conserved. The failure ofmost virus seropositive specimens to react with the amino terminal halfof gp120 represented by env-1 may indicate that there are not suchhighly conserved immunodominant sequential epitopes within this region.These data suggest that either the amino terminal region of gp120 is apoor immunogen in humans, or humans respond in a strain-specific fashionso that only those individuals infected with a highly homologous virusare detected with env-1 from ARV-2. Another explanation centers aroundthe possibility that conformational epitopes may be a feature of theN-terminal portion of gp120; immunoblotting would miss antibodiesdirected at conformational determinants. Another possible explanation isthat the immunogenicity of the amino terminal region of gp120 is maskedby glycosylation or by structural constraints due to association ofgp120 with gp41 and/or the viral membrane.

It was also found that all sera reacting in immunoblots with env-3 alsoreacted with the fusion protein env-5b. In fact, several sera that wereonly weakly positive on env-3 were clearly positive on env-5b. Thesensitivity of an immunoblot assay is limited by the amount of antigenthat is present in the preparation. Since env-5b expression levels werehigh compared to the levels of expression of env-3 in yeast (env-5brepresented approximately 2-5% of the total bacterial protein, whereasenv-3 was less than 0.1% of the total yeast protein), it was notunexpected the env-5b immunoblot assays were more sensitive than theenv-3 immunoblot assays.

6.2 ELISA with Env Polypeptides

To provide a quantitative assessment of the antibody response detectedusing these recombinant polypeptides representing regions of twosubunits of the HIV envelope glycoprotein complex, env recombinantpolypeptides (Section 4.2) were purified and enzyme-linked immunosorbentassays (ELISA) configured.

Test sera were the same as described in Section 6.1. The ELISAprocedures were modifications of those described previously. SeeBrun-Vezinet et al. (1984) Lancet I:1253-1256; Saxinger et al. (1983)Lab. Invest. 49:371-377; Weiss et al. (1985) J.A.M.A. 253:221-225;Steimer et al. (1986) J. Virol. 58:9-160.

For virus ELISAs, microtiter plates (Dynatech, Immulon I) were coatedwith 5 μg/ml of SDS-disrupted sucrose gradient purified ARV-2 virus.Human serum samples, diluted 1/100, were added to the wells and theplates incubated at 37° C. After 1 h, the plates were washed and goatantiserum to human immunoglobulin conjugated with horseradish peroxidase(Cappel Laboratories) and diluted 1/4000 was added to the wells. Theplates were incubated for 30 min. at 37° C., washed and the substratesolution (150 μg/ml, 2,2′-azino-di-3′-ethylbenzylthiazoline sulfate in0.1 M citrate, 0.001% H₂O₂, pH 4.0) was added for 30 minutes. The plateswere read on a ELISA reader at 415 nm with a reference wavelength of 600nm. Samples were scored as positive when their assay result was greaterthan five times the absorbance obtained with the negative control poolednormal human serum (NHS).

For recombinant env polypeptide ELISAs, microtiter plates were coatedwith 2 μg/ml of purified env-2 or env-5b (Section 7.1). Serum sampleswere assayed for antibodies to these antigens by the procedure describedabove. Included in the serum diluent for the env-2 antibody ELISA wasincluded extract (final concentration 100 μg/ml) from yeast transformedwith the vector alone lacking the env-2 insert. Similarly, 100 μg/ml ofan extract from untransformed E. coli was added to the diluent for theenv-5b antibody ELISA.

FIG. 16 (panel A) shows the results of a titration of antibodies in anAIDS patient's serum that reacted with env-2. Pooled normal human serum(NHS) (0) and serum 0036 from an AIDS patient ( ) were diluted 1/25 andthen by serial 2-fold dilutions. The titer of env-2 antibodies in thisparticular serum specimen was approximately 1400. The signal with thecontrol sample (NHS) was low at all dilutions.

Env-5b was purified and used as antigen in an ELISA for antibodies tothe gp41 polypeptide. FIG. 16 (panel B) shows the results of a titrationof env-5b antibodies in the same serum sample that was titeredpreviously in the env-2 ELISA. No appreciable signal was seen with NHS.

Serum samples from 88 blood donors, all seronegative in an ELISA forAIDS retrovirus antibodies were tested at a dilution of 1/100 in theenv-2 and env-5b antibody ELISAs (FIG. 17). The average signal in theenv-2 antibody ELISA was 0.045±0.033 with a range of 0 to 0.149. In theenv-5b ELISA these same sera yielded an average signal of 0.034±0.030with a range of 0 to 0.144. The cut-off for both assays was set at fivetimes the average signal of these 88 seronegative specimens. For theenv-2 antibody ELISA the cut-off was 0.225, and for the env-5b assay itwas 0.170.

The results of the env-2 and env-5b ELISAs with the same panel of virusseropositive specimens described previously (see the table in Section7.1) are presented in FIG. 18. The dotted line in the top paneldesignates the cut-off (0.174) in the env-2 ELISA. The dotted line inthe bottom panel (0.154) is the cut-off in env-5b antibody ELISA. Eachdata point is the average of duplicate assays. All of the sera,regardless of diagnosis, yielded the maximum signal in the env-5bantibody ELISA.

Serum antibodies were clearly detected in the env-2 ELISA with specimensfrom 20 of the 21 contacts, 24 of the 25 ARC patients, and 35 of the 38AIDS patients. The remaining five sera, one from a contact, one from anARC patient and three from AIDS patients, are considered to beborderline. The contact and ARC sera scored just below the assay cut-offwhile the three AIDS sera scored just above the cut-off. These same serawere all clearly positive in the env-5b antibody ELISA.

FIG. 19 compares antibody titers of the above sera for both env-2 andenv-5b. These serum samples were assayed in the env-2 and env-5b ELISAsat dilutions ranging from 1/100 to 1/51,200. The titers reported are thedilution at which half-maximum absorbance was attained. The env-2 titersare shown as dark circles, and the env-5 titers are open circles. Thehorizontal lines show the mean titers of sera in each group. Borderlinesera (less than 0.15 above assay cut-off) are shown as “±”. Sera withtiters below 1/100, but not borderline, are plotted as having titers of1/100.

Antibody titers in sera obtained from all three groups were higher toenv-5b than to env-2). The average titer of env-2 antibodies in seraobtained from contacts was 2224 (range <100-7975) compared to an averagetiter of env-5b antibodies of 8988 (range 650-25,000). Among the ARCpatients the average env-2 and env-5b antibody titers were 2480 (range<100-12,800) and 12,560 (range 42062,000), respectively. Finally, forAIDS patients the average env-2 antibody titer was 1394 (range<100-12,800) compared to an average env-5b antibody titer of 7059 (range<100-42,000).

The ratio of env-5b to env-2 antibody titers was 5.06, 4.04 and 5.06 forcontacts, ARC patients and AIDS patients respectively. Since theseratios remain constant, regardless of diagnosis, it is unlikely thatthere is a selective decline in the antibody titer to one or the otherof the env polypeptides with progressive disease. Instead, it indicatesthat individuals with low titer antibodies to one env polypeptide tendto have low titers of antibodies to the other polypeptide. Examining theenv-5b antibody titers of those individuals with low (<200) titers ofenv-2 antibodies (table below) supported this conclusion; these sera allhad correspondingly low env-5b antibody titers.

Serum Clinical Env-2 Antibody Env-5b Antibody Number Group Titer Titer4687 C* <100 1300 4623 ARC 115 3100 4648 ARC 125 1000 4672 ARC <100 5904677 ARC <100 450 4685 ARC 170 420 4616 AIDS <100 430 4619 AIDS 100 9004620 AIDS 130 620 4625 AIDS <100 1500 4626 AIDS 100 4300 4627 AIDS 100200 4631 AIDS 170 500 4637 AIDS 190 480 4653 AIDS <100 2500 4654 AIDS100 400 4660 AIDS 100 760 4664 AIDS 155 300 4667 AIDS 100 680 *Contacts.6.3 ELISA with Gag Polypeptides

Large-scale purification of p25gag from bacterial extracts (Section3.1.5) provided sufficient antigen for a survey of p25gag seropositivityamong various risk and patient groups. These sera were tested first in avirus ELISA to identify those with antibodies to the virus and then inthe p25gag ELISA to determine the proportion of virus seropositiveindividuals with antibodies to p25gag.

Ninety-six sera from random blood donors, all of whom were negative forAIDS virus antibodies in the virus ELISA, scored negative in the ELISAfor p25gag antibodies. The average signal in the p25gag ELISA of thesesera was 0.034±0.016 optical density units (OD) with a range of 0.011 to0.089 (not shown).

A panel of 100 sera was then examined, consisting of 28 specimens fromhigh-risk individuals with no symptoms of the disease, but withpotential exposure to the virus through sexual contact with AIDSpatients (contacts), 33 sera from patients with AIDS-related complex(ARC), and samples from 39 patients with AIDS. Eighty-six of the 100specimens were positive in the virus ELISA, an indication that theseindividuals had mounted an immune response to one or more viralantigens. The number of virus ELISA positive contacts was 21 (75%), ARCpatients was 27 (81.8%), and AIDS patients was 38 (97.4%). The resultsof the p25gag antibody ELISA of these specimens are summarized in thetable below. Of the 86 sera from the total panel that were positive inthe virus ELISA, only 34 (39.5%) scored positive in the p25gag ELISA.When the results were grouped according to diagnosis, however, thecontacts had the highest number of virus seropositive individuals withantibodies to p25gag (71%), the ARC patients were intermediate (48%),and the AIDS patients were the lowest (16%). Also tested were the 14virus ELISA negative specimens from this panel of 100 sera in the p25gagELISA, and all scored negative (not shown).

The data presented in the table suggest that with an increase in theseverity of the disease, there is a decline in the proportion of virusseropositive individuals with antibodies to p25gag. To determine thesignificance of the observed differences in p25gag seropositivitybetween groups, we used an X² test with pairwise comparison of groups.This analysis shows that the probability that the observed differencesin frequency of p25gag seropositivity between the contacts and AIDSpatients could have been due to chance was less than 1 in 2000.Similarly, the probability that the difference between ARC and AIDSpatients was due to chance was very small (1 in 200). A comparison ofcontacts and ARC patients, however, gave a higher probability (1 in 10)that the observed difference was due to chance. A larger sample sizewould be required to establish the significance of this difference withgreater certainty.

These results suggest that monitoring p25gag seropositivity ofindividuals infected with the AIDS retrovirus might be of diagnosticvalue. Although patients with more severe forms of the disease clearlyhave antibodies to other viral antigens, such as the envelopeglycoproteins [Barin et al. (1985) Science 228:1097; Montagnier et al.(1985) Virology 144:283], they are less likely than those in earlierstages to have antibodies to p25gag.

The above results were obtained with p25gag antigen produced in E. coli.Similar results have been seen with the antigen produced in yeast.

p25gag Antibody ELISA of Virus Seropositive Specimens^(a) NumberCategory or diagnostic scoring percentage^(b) (number in group) positivepositive Contacts (21) 15 71 ARC patients (27) 13 48 AIDS patients (38)6 16 total (86) 34 39 ^(a)Samples were categorized as virus seropositiveif they yielded greater than five times the signal obtained with normalhuman serum (NHS) in a virus ELISA assay using disrupted ARV-2 virus asantigen. The p25gag antibody ELISA procedure was as follows: ELISAplates were coated with 5 μg/ml of p25gag purified from bacterialextracts. In the diluent was included a lysate from untransformed E.coli. All were diluted 1/100 for assay. The conjugate was horseradishperoxidase conjugated goat antiserum to human immunoglobulin (Cappel,No. 3201-0081). ^(b)A sample was scored as positive when the average ODreading was greater than five times the signal with pooled normal humanserum (NHS). In this particular assay this was OD₄₁₄ = 0.200.6.4. Western and ELISA with Pol Polypeptides

A panel of 10 sera that scored positive in the ELISA for viralantibodies with disrupted virus as the antigen was selected to comparethe immunoreactivity of viral p31 with those of the two recombinantproteins. This panel included eight sera that were positive and two serathat were negative for antibodies to viral p31 in virus immunoblots.Lysates of pTP31.2 (Section 3.4.2) and pTSp31 (Section 3.5)transformants were electrophoresed and electroblotted, and strips of theblots were reacted with individual serum samples (see table below). Theeight sera that were positive on viral p31 also reacted with the 30- and48-kDa bands in lysates of bacterial cells containing pTP31.2(expressing p31pol) and pTSp31 (expressing SOD-p31pol fusion),respectively. The two sera that were negative on viral p31 were alsonegative on both of the recombinant proteins. Pooled normal human serumwas negative in all three Western blot assays.

The SOD-p31 fusion protein was purified and used as a source of antigenfor an ELISA for testing sera for antibodies to p31. Prior to decidingto pursue the fusion protein as a source of antigen for the assay, serafrom 300 random blood donors and a panel of 100 sera obtained fromhigh-risk, AIDS, and ARC patients was screened for antibodies topurified human SOD in an ELISA. None of these sera scored positive (datanot shown).

The ELISA protocol was as follows: Microtiter plates were coated with 2μg/ml of SOD-p31 in borate buffer (pH 9). In the diluent was includedextract from E. coli (pTAC7). This extract was necessary to absorb outantibodies in human sera that reacted with minor contaminants in thepurified SOD-p31. The ELISA protocol from this point was identical tothe procedure described for other recombinant HIV antigens describedabove.

A panel of sera from 100 consecutive blood donors that were allseronegative in the virus ELISA was tested in the p31 ELISA. These seraall scored very low in the assay (FIG. 31 a). The average ELISA resultfor these sera when they were assayed at a 1/100 dilution was 0.026 witha range of 0.001 to 0.117 and a standard deviation of 0.012. The resultsobtained with 85 virus-seropositive samples are presented in FIG. 31 b.The ELISA results with sera that did not react with the p31 band in thevirus Western blot assay are indicated by shading. In general, sera thatwere positive in virus Western blots for p31 antibodies were clearlypositive in the p31 antibody ELISA. There were, however, three sera thatdid not react with p31 in virus immunoblots that were clearly positivein the p31 ELISA.

Three possible explanations for viral p31 immunoblot-negative serascoring positive in the p31 ELISA were considered. First, the ELISA mayhave been more sensitive than the virus immunoblot assay for detectingantibodies to p31. Second, these sera may have scored positive in theELISA owing to immunological reactivity with E. coli proteinscontaminating the SOD-p31 preparation that were not absorbed out by thebacterial lysate in the antibody diluent. Third, these sera may havescored positive in the ELISA because they contained antibodies to theSOD portion of the fusion protein.

To clarify this, these three sera were tested on blots of directlyexpressed p31 by using the pTP31.2 lysate, which should contain a muchhigher concentration of p31 antigen than virus. All three of these serareacted with the p31 species in this assay (data not shown). Thus, thesesera scored positive in the ELISA because of its greater sensitivitythan virus immunoblots for detecting p31 antibodies, not because ofreaction with E. coli contaminants or SOD.

We also tested the remaining eight viral p31 Western blot-negative serain immunoblot assays of pTP31.2 lysates. The five sera scoring lowest inthe ELISA (FIG. 31 b) failed to react with p31 in this assay. However,the three remaining sera, scoring between 0.15 and 0.3 in the ELISA,reacted in immunoblots with directly expressed p31 (data not shown).Thus, we concluded that of this panel of 85 seropositive samples, 80(95%) had detectable antibodies to the p31 antigen.

These data clearly demonstrate the utility of the HIV pol endonucleasepolypeptide in serodiagnosing HIV infection. It should be noted thatserological results were identical when SOD-p31 produced in yeast wasused instead of the bacterially expressed protein.

Comparison of Western blot results of various virus-seropositive samplesin viral p31, recombinant p31 expressed directly in E. coli and as afusion protein with human SOD Reactivity with: Recombinant Serum Viralp31 Recombinant p31 SOD-p31 4607 + + + 4608 + + + 4620 + + + 4625 + + +4626 + + + 4642 − − − 4643 + + + 4646 + + + 4659 − − − 0036 + + +NHS^(b) − − − ^(a)Strips from immunoblots of electrophoresed virus,pTP31.2 extracts, and pTS31 extracts were reacted with a 1/100 dilutionof each serum. ^(b)NHS, pooled normal human sera obtained from MedicalSpecialities Laboratories.7. HIV Immunization

Recombinantly produced viral envelope protein was used to generateanti-HIV antibodies in experimental animals which were capable ofneutralizing the infectivity of the virus. The purified env-2 (Section4.2.2) was injected into both mice and guinea pigs and the sera hadneutralizing activity in an in vitro neutralization assay.

The HIV neutralization assay that was developed measures the ability ofserum specimens to neutralize directly the infectivity of HIV in atissue culture system (described in commonly owned, co-pending U.S.patent application No. 946,539, the disclosure of which is herebyincorporated by reference). Diluted sera are mixed with an equal volumeof virus inoculum, the mixture is incubated for 30 minutes at roomtemperature and then 0.1 ml of the mixture is added to a 1 ml culture ofpermissive HUT-78 cells (1×10⁴ cells/ml) in 24-well microtiter plates.Seven days later the cells are harvested, lysed with 0.1% Triton X-100in PBS.

Infection is monitored by measuring the levels of intracellular p25gagantigen with a capture enzyme-linked immunosorbent assay (ELISA) Steimeret al. (1986) virology 150:283-290. This assay uses a murine monoclonalantibody immobilized on the assay plate to capture p25gag and a rabbitpolyclonal antiserum as the detecting reagent. The virus inoculum thatwas used for all strains of HIV was adjusted to yield 40-80 ng/ml ofintracellular p25gag and approximately 10% of the HUT-78 cells in theculture were infected under these conditions.

7.1. Env-2 Immunization of Mice

In order to test if env-2 polypeptide derived from yeast was capable ofeliciting neutralizing antibodies, mice were immunized with env-2(Section 4.2.2).

Three Balb/c mice were injected three times with 10 μg of env-2 in alumat two week intervals. The mice were bled one week following the thirdinjection and their sera tested for env-2 antibodies in ELISA. Normalmouse serum from a pool of sera obtained from unimmunized Balb/c mice,was included as a control. The sera was diluted 1:10, and by serial2-fold dilutions, and reacted in the env-2 ELISA. All three mice hadmounted an antibody response to the injected env-2 (see below). The serawere then tested in the neutralization assay at a 1:20 dilution. Serafrom two of the immunized mice showed significant neutralization of HIVat a 1:20 dilution (see below).

Neutralization activity Serum Env-2 (percent sample antibody titerneutralization) normal <10 18 mouse 1 420 97 mouse 2 230 4 mouse 3 200767.2. Env-2 Immunization of Guinea Pigs

Twelve Hartley guinea pigs were used to study the effectiveness of theenv-2 polypeptide (Section 4.2.2) in eliciting neutralizing antibodies.The sera were also tested for their ability to neutralize differentstrains of the HIV virus.

Six of the guinea pigs were immunized with 50 μg of antigen withadjuvant (see below) and six were immunized with the adjuvant aloneaccording to the following protocol:

Adjuvant: contains 0.5 mg Monophosphoryl Lipid A (MPL), 0.5 mg TrehaloseDimycolate (TDM), 0.5 mg Cell Wall Skeleton (CWS) (isolated fromattenuated tubercle bacillus: Bacillus-Calmette-Guerin) lyophilized in40 μl of oil (Squalene) and 0.2% Tween 80 in water (adjuvant obtainedfrom Ribi Immunochem Research, Inc., Hamilton, Montana). Vaccine:Reconstitute the adjuvant by adding 600 μl of PBS and vortexingvigorously for 2-3 minutes. Mix 300 μl of the adjuvant with 300 μg ofthe env-2 polypeptide and PBS to bring total volume to 600 μl. Vortexvigorously for 2-3 minutes. Inject each guinea pig with 100 μl ofvaccine in foot pad. Control: Dilute remaining 300 μl of adjuvant with300 μl of PBS and vortex vigorously for 2-3 minutes. The six controlguinea pigs are injected with 100 μl each in foot pad. Immunization: Twoto four days prior to immunization Schedule: obtain “prebleed” bloodsample for guinea pigs: Day 0: Primary immunization; Day 21: secondimmunization; Day 28: obtain first blood sample; Day 42: thirdimmunization; Day 49: obtain second blood sample.

RESULTS

ELISA Titers² against env-2 Neutralization Titer² Bleed 1 Bleed 2 ofBleed 2 Immunized Guinea Pigs¹ A 81 n.d. n.d. B 171 605 <10 C 111 n.d.n.d. D 510 1,274 <10 E 501 10,756 220 F n.d. 4,050 40 Control GuineaPigs¹ A′ <25 <25 <10 B′ <25 <25 <10 C′ <25 <25 <10 D′ <25 <25 <25 E′ <25<25 n.d. F′ <25 <25 n.d. n.d. = not determined ¹sera was heatinactivated ²dilution that gives 50% of inhibition of viral inoculum(ARV-2)The sera from guinea pigs E and F were tested for their ability toneutralize various isolates of HIV which have been associated with AIDS.

Neutralizing Neutralizing HIV Titer of Guinea Titer of Guinea IsolatePig E, Bleed 2 Pig F, Bleed 2 LAV¹ 70 30 ARV-2² 220 40 ARV-3³ <10 n.d.ARV-33³ <10 n.d. HIV-Zr6⁴ <10 n.d. ¹Barre-Sinoussi et al. (1983) Science220:868-871. ²Levy et al. (1984) Science 225:740-842. ³Staben et al.(1986) in: Vaccines 86, Cold Spring Harbor Laboratory, New York, pp.345-350. ⁴Srinivassen et al. (1987) Gene 52:71-82.8. Deposits of Biological Materials

Samples of organisms that contain HIV clones or express theabove-described hTLR polypeptides were deposited at the American TypeCulture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. underthe provisions of the Budapest Treaty. The accession numbers and datesof these deposits are listed below.

ATCC Deposited Material Accession No. Deposit Date λ-ARV-2(7D) 40143 26Oct. 1984 λ-ARV-2(8A) 40144 26 Oct. 1984 λ-ARV-2(9B) 40158 25 Jan. 1985E. coli HB101 (pSV7c/env) 67593 23 Dec. 1987 E. coli HB101(pCMV6ARV120tpa) — — E. coli D1210 53246 27 Aug. 1985 (pGAG25-10) E.coli D1210 — — (pSOD/env5b) E. coli D1210 — — (pTP31.2) E. coli D1210(pII-3) 67549 28 Oct. 1987 Saccharomyces cere- 20768 27 Aug. 1985 visiaeP017 (pC1/1 (pSP31-GAP-ADH2) Saccharomyces cere- 20769 27 Aug. 1985visiae 2150 (pDPC303) Saccharomyces cere- 20827 23 Dec. 1986 visiae2150-2-3 (pAB24-GAP-env2) Saccharomyces cere- — — visiae 2168(pBS24/SOD-SF2env4) Saccharomyces cere- — — visiae AB110(pC1/1-p25-ADH-GAP) Saccharomyces cere- — — visiae AB116(pBS24.1/SOD-SF2env4-5) Saccharomyces cere- — — visiae (pAB24/RT4)Saccharomyces cere- — — visiae JSC302 (pC1/1-GAP-p53)

These deposits are provided for the convenience of those skilled in theart. These deposits are neither an admission that such deposits arerequired to practice the present invention, nor that equivalentembodiments are not within the skill of the art in view of the presentdisclosure. The public availability of these deposits is not a grant ofa license to make, use or sell the deposited materials under this or anyother patent. The nucleic acid sequences of the deposited materials areincorporated in the present disclosure by reference and are controllingif in conflict with any sequence described herein.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that changes and modifications may bepracticed within the scope of the appended claims.

1. In an immunoassay to detect the presence of antibodies to a humanimmunodeficiency virus (HIV) in a human sample comprising contactingsaid sample with an envelope (env) antigen comprising an amino acidsequence from the env domain of said HIV and determining whetherantibodies are bound to said env antigen, the improvement comprisingemploying as said env antigen a synthetic polypeptide comprising anantigenic amino acid sequence from the env domain of HIV as shown inFIG.
 4. 2. A method of detecting antibodies to a human immunodeficiencyvirus (HIV) in a human sample, comprising: a) providing a solid supporthaving bound thereto a synthetic polypeptide comprising an envelope envantigen comprising an antigenic amino acid sequence of the env domain ofHIV as shown in FIG. 4, wherein said env antigen is a syntheticpolypeptide; b) contacting said solid support with said human sample toprovide a sample-contacted support; c) washing said sample-contactedsupport to provide a washed support; and d) determining whether humanantibodies are bound to said washed support.
 3. The method of claim 1wherein said human sample comprises serum.
 4. The method of claim 2wherein said human sample comprises serum.
 5. The method of claim 1wherein step (d) comprises contacting said washed support with labeledantibodies to human Ig and the specific binding of said labeledantibodies to said washed support is measured.
 6. The method of claim 2wherein step (d) comprises contacting said washed support with labeledantibodies to human Ig and the specific binding of said labeledantibodies to said washed support is measured.
 7. An article ofmanufacture adapted for use in an immunoassay for antibodies to a humanimmunodeficiency virus (HIV) comprising a solid support having boundthereto to synthetic polypeptide comprising at least a portion of anenvelope (env) antigen comprising an antigenic amino acid sequence ofthe env domain of said HIV, wherein said env antigen is a syntheticpolypeptide comprising an antigenic amino acid sequence from the envdomain of HIV as shown in FIG.
 4. 8. The immunoassay of claim 1 whereinsaid env antigen contains at least 15 amino acid residues.
 9. The methodof claim 2 wherein said env antigen contains at least 15 amino acidresidues.
 10. The article of manufacture of claim 7 wherein said envantigen contains at least 15 amino acid residues.